• Journal of Crop Science and Biotechnology
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• v.11 no.4
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• pp.233-236
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• 2008

A positive selectable marker system was adapted for transformation of mature embryo-derived calli of Indica rice (Oryza sativa L.) utilizing the PMI gene encoding for phosphomannose-isomerase that converts mannose-6-phosphate to fructose-6-phosphate. The transformed cells grew on medium supplemented with 3% mannose as carbon source and calli were selected on media containing various concentrations of mannose. Molecular analyses showed that the transformed plants contained the PMI gene. The results indicate that the mannose selection system can be used for Agrobacterium-mediated transformation of mature embryo in rice to substitute the use of conventional selectable markers in genetic transformation.

• Journal of Plant Biotechnology
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• v.3 no.1
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• pp.19-25
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• 2001

Induction of somatic embryogenesis from Brazilian eggplant variety F-100 was studied in response to four auxin types. NAA, at the optimal concentration of 54 $\mu\textrm{m}$, was the only one that resulted in the induction of somatic embryos in either leaf and cotyledon explant and, at murk lower intensity and frequency, in hypocotyl and epicotyl explants. The optimal temperatures for embryo induction were 28 and 35$^{\circ}C$ for cotyledon and leaf explants. Incubation at 22$^{\circ}C$ caused a significant reduction both in the frequency and intensity of induction. This system was used to study the effects of position and orientation of the tissue on the culture medium as well as of antibiotics and explant co-cultivation with Agrobacterium on the efficiency of somatic embryo induction. The intensity of embryo induction was greater in the midsections of cotyledons relative to apical and basal regions, when the abaxial surface was in contact with the culture medium. The presence of antibiotics resulted in approximately 40-60% reduction of embryo induction relative to control explants, which originated 335$\pm$26.6 embryos. Co-cultivation with Agrobacterium before treatment with antibiotics caused a more drastic reduction (80-99%). Ampicilin treatment after cocultivalion with Agrobacterium caused the least inhibitory effect, allowing the production of 60 embryos/explant.

• Plant Biotechnology Reports
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• v.4 no.2
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• pp.139-148
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• 2010

Jatropha curcas L. (Physic nut) is a commercially important non-edible oil seed crop known for its use as an alternate source of biodiesel. In order to investigate the morphogenic potential of immature embryo, explants from four developmental stages were cultured on medium supplemented with combinations of auxins and cytokinins. It was found that the size of embryo is critical for the establishment of callus. Immature embryos (1.1-1.5 cm) obtained from the fruits 6 weeks after pollination showed a good response of morphogenic callus induction (85.7%) and subsequent plant regeneration (70%) with the maximum number of plantlets (4.7/explant) on Murashige and Skoog's (MS) medium supplemented with IBA (0.5 $mg\;l^{-1}$) and BA (1.0 $mg\;l^{-1}$). The above medium when supplemented with growth adjuvants such as 100 $mg\;l^{-1}$ casein hydrolysate + 200 $mg\;l^{-1}$ L-glutamine + 8.0 $mg\;l^{-1}$ $CuSO_4$ resulted in an even higher frequency of callus induction (100%). Plant regeneration (90%) with the maximum number of plantlets (10/explant) was achieved on MS medium supplemented with 500 $mg\;l^{-1}$ polyvinyl pyrrolidone + 30 $mg\;l^{-1}$ citric acid + 1 $mg\;l^{-1}$ BA + 0.5 $mg\;l^{-1}$ Kn + 0.25 $mg\;l^{-1}$ IBA. It was observed that plantlet regeneration could occur either through organogenesis of morphogenic callus or via multiplication of pre-existing meristem in immature embryos. The age of immature embryos and addition of a combination of growth adjuvants to the culture medium appear to be critical for obtaining high regeneration rates. Well-developed shoots rooted on half-halfstrength MS medium supplemented with 0.5 $mg\;l^{-1}$ IBA and 342 $mg\;l^{-1}$ trehalose. The rooted plants after acclimatization were successfully transferred to the field in different agro-climatic zones in India. This protocol has been successfully evaluated on five elite lines of J. curcas.

• International Journal of Oral Biology
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• v.42 no.3
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• pp.107-115
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• 2017

Human malignant melanoma is an aggressive skin cancer which has been rising at a greater rate than any other cancers. Although various new therapeutic methods have been developed in previous studies, this disease has properties of high proliferation and metastasis rate which remain obstacles that have lead to a poor prognosis in patients. It has been reported that a specific Lactobacillus extract has anti-cancer and -metastasis effect in vitro and in vivo. However, previous research has not specified precisely what effect the Lactobacillus rhamnosus GG (LGG) extract has had on human malignant melanomas. In this study, we showed that the LGG extract has anti-cancer and -metastasis effects on the human malignant melanoma cell lines, A375P and A375SM. At first, it was found that, while the LGG extract affects human neonatal dermal fibroblasts slightly, it induced the dose-dependent anti-cancer effect on A375P and A375SM by a WST-1 proliferation assay. As a result of a real-time PCR analysis, the expression patterns of several genes related to cell cycle, proliferation, and apoptosis were modulating in a manner that inhibited the growth of both malignant melanoma cell lines after the treatment of the LGG extract. Furthermore, genes related to the epithelial-mesenchymal transition were down-regulated, and migration rates were also decreased significantly by the LGG extract. Our study showed that the LGG extract could be used as a potential therapeutic source.

• Korean Journal of Medicinal Crop Science
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• v.7 no.1
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• pp.1-6
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• 1999

Basic informations for direct somatic embryo formation in Rehmannja glutinosa Lib. were obtained in 500ml erlenmyer flask. The ratio of ammonium to nitrate nitrogen of 825(mg/l) : 1900(mg/l) was proper condition for somatic embryo formation from stem and petiole explants and 3% sucrose was the most effective carbon source. Full strength MS medium with 2mg/l BA was better than LS medium for somatic embryogenesis. The initial pH 5.7 of medium(full strength MS with 2.0mg/l BA and 0.1mg/l NAA) was good for embryo production. Potassium ion was taken up rapidly within 2 weeks. while $Ca^{++}$ and $Mg^{++}$ ion contents were almost constant during culture period. Sucrose hydrolysis occurred throughout the culture, while glucose and fructose were absorbed simultaneously from the third week of culture.

• Journal of Embryo Transfer
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• v.23 no.3
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• pp.223-227
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• 2008

The cryopreservation of Hanwoo embryos has become an integral part of assisted reproduction in animal. The objective of this study was to assess the effect of The objectives of this study were: (1) to evaluate the influence of bovine embryo developmental stage on in vitro embryo development after freezing, (2) to study the efficiency compared with conventional freezed embryos at different embryo source. For conventional slow-freezing, day 7 or 8 expanded blastocysts were collected. The standard freezing medium was 1.8 M ethylene glycol (EG). Embryos were equilibrated in 1.8 Methylene glycol(EG) with 0.1 M sucrose in Dulbecco's phosphate-buffered saline (D-PBS) supplemented with 0.5% bovine serum albumin. Embryos were then loaded individually into 0.25 ml-straw and placed directly into cooling chamber of programmable freezer precooled to $-7^{\circ}C$, after 2 min, the straw was seeded, maintained at $-7^{\circ}C$ for 8 min, and then cooled to $-35^{\circ}C$ at $0.3^{\circ}C$/min, plunged and stored in liquid nitrogen for at least 3 days. For thawing, the straw containing embryos were warmed in air for 10 see and exposed to $37^{\circ}C$ water for 20 sec. Straws were then removed from $37^{\circ}C$ water. Rates of blastocyst survive and hatched were evaluated at 12 to 48h post-warming. The re-expansion and hatched rates of morula embryos were significantly lower than those obtained for blastocysts and expansion blastocysts (31.6%, 10.5% vs, 68.9%, 22.2% vs, 73.7%, 53.6%, respectively). No differences in re-expansion rates were found between in vivo and in vitro blastocysts. whereas hatched rates was significantly higher (51.2%) in vivo compared with in vitro embryos (18.6%). in conclusion, demonstrate that conventional freezing can be used successfully in cryopreservation of in vitro and in vivo bovine embryos, and that it might be considered for use in commercial programs and embryo preservation.

• Journal of Embryo Transfer
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• v.13 no.3
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• pp.235-243
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• 1998

In this study, we investigated the effects of three kinds of culture medium (Charles and Rosenkrans; CRlaa, Tyrode's; TALP, synthetic oviduct fluid: SOF), insulin transferrin + selenium complex (ITS), macromolecules(polyvinyl alcohol: PVA, fetalb-ovine serum: FBS) and NaCl on the development of early bovine embryos. In experiment 1, there were no differences in embryo development among three kinds of embryo culture medium (CR $l_{aa}$ , TALP, SOF). In experiment 2, BSA, FBS and PVA were added each in TALP as macromolecule sources. The developmental rates of embryos in BSA or FBS added TALP were significantly higher than in PVA added one (p〈0.01), but there was no difference between BSA and FBS added groups. In experiment 3, bovine embryos were cultured in TALP with the following supplements: BSA alone(1, 3 or 8 mg/ml, each) or BSA(1, 3 or 8 mg/ml, each)+ITS (10$\mu\textrm{g}$/m1 insulin, 5 $\mu\textrm{g}$/ml transferrin, 5 ng/ml selenium). In higher concentration of BSA and ITS supplemented groups, the developmental rates over compacted morula were higher than others, but there was a significant effect of ITS only in 1 mg/ml of BSA added group (p〈0.05). In experiment 4, the effect of reduced concentration of NaCl was evaluated. The developmental rate over compacted morula in the medium containing 90 mM of NaCl was higher than in 114 mM group (p〈0.05). In conclusion, BSA could be used as a macromolecule source in bovine embryo culture, and ITS, as a serum substitute, could be used for improving of embryonic development. Also, reduction of NaCl concentration from 114 mM to 90 mM may improve the development of bovine embryos.bryos.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.1
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• pp.1-7
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• 2000

Objective: Mammalian embryos undergo changes of energy environment for transfer from oviduct to uterus. Also, the human reproductive organ (oviduct, uterus) contains energy sources of different concentration (oviduct - glucose: 0.5 mM, pyruvate: 0.32 mM, lactate: 10.5 mM; uterus - goucose: 3.15 mM, pyruvate: 0.1mM, lactate: 5.87 mM, respectively). This study was conducted to examine the effect of these energy sources added in DMEM with glutamine on the mouse embryo development. Methods: There was used ICR female mouse. Two cell embryos of mouse are collected by method of 'flushing'. Flushing fluid was used Ham's F-10 added to 20% FBS. The collected 2 cell embryos were cultured in media such as Control (only DMEM), group A and B (DMEM supplemented with 0.5 mM and 3.15 mM glucose), and group C and D (DMEM supplemented with 0.1 mM and 0.32 mM pyruvate), and group E and F (DMEM supplemented with 5.87 mM and 10.5 mM lactate). All experimental media supplemented with 20% hFF, respectively. Pattern of embryo development was observed to interval at 24hr during 96hr. Results : The media with glutamine added glucose (group A: 51.0%; group B: 48.4%) was significantly (p<0.05) higher than other experimental group in development into the morula stage after 24 hr in culture, but not significantly different compared with control and the rate of development into the blastocyst was significantly (p<0.05) low in the both of pyruvate (group C: 7.9% group D: 6.8%) and lactate (group E: 7.1%, group F: 7.1%) treatment group after 48 hr in culture. Development into the blastocyst and hatched balstocyst after 72 hr in culture revealed similarly in control (81.9%) and glucose treatment group (group A: 83.3%, group B: 82.8%). However, development into the hatched and attached blastocyst after 96hr in culture revealed significantly (p<0.05) development in the glucose treatment group (group A: 82.3%, group B: 78.5%) than control (63.2%), and its of pyruvate (group C: 34.1%, group D: 34.1%) and lactate (group E: 25.9%, group F: 33.3%) treatment group were significantly (p<0.05) lower than control similar to previous observations. Conclusion : The glucose added to the DMEM with only glutamine, as energy source, was highly to the rate of development compared with control, but the other energy sources were not, synthetically. Above refer to, the human reproductive organ (oviduct, uterus) contains energy sources of different concentration. Thus, further studies are will examine continuously to effects by interaction of different energy sources in the mouse embryo development, and these results will provide to foundation on the human embryo culture.

• Journal of Embryo Transfer
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• v.27 no.3
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• pp.175-181
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• 2012

The synovial tissues are a valuable MSCs source for cartilage tissue engineering because these cells are easily obtainable by the intra-articular biopsy during diagnosis. In this study, we isolated and characterized the canine MSCs derived from synovial fluid of female and male donors. Synovial fluid was flushed with saline solution from pre and post-puberty male (cM1-sMSC and cM2-sMSC) and female (cF1-sMSC and cF2-sMSC) dogs, and cells were isolated and cultured in advanced-DMEM (A-DMEM) supplemented with 10% FBS in a humidified 5% $CO_2$ atmosphere at $38.5^{\circ}C$. The cells were evaluated for the expression of the early transcriptional factors, such as Oct3/4, Nanog and Sox2 by RT-PCR. The cells were induced under conditions conductive for adipogenic, osteogenic, and chondrogenic lineages, then evaluated by specific staining (Oil red O, von Kossa, and Alcian Blue staining, respectively) and analyzed for lineage specific markers by RT-PCR. All cell types were positive for alkaline phosphatase (AP) activity and early transcriptional factors (Oct3/4 and Sox2) were also positively detected. However, Nanog were not positively detected in all cells. Further, these MSCs were observed to differentiate into mesenchymal lineages, such as adipocytes (Oil red O staining), osteocytes (von Kossa staining), and chondrocytes (Alcian Blue staining) by cell specific staining. Lineage-specific genes (osteocyte; osteonectin and Runx2, adipocytes; PRAR-${\gamma}2$, FABP and LEP, and chondrocytes; collagen type-2 and Sox9) were also detected in all cells. In this study, we successfully established synovial fluid derived mesenchymal stem cells from female and male dogs, and determined their basic biological properties and differentiation ability. These results suggested that synovial fluid is a valuable stem cell source for cartilage regeneration therapy, and it is easily accessible from osteoarthritic knee.

• Journal of Embryo Transfer
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• v.33 no.2
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• pp.85-97
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• 2018

The trans-differentiation potential of mesenchymal stem cells (MSCs) is employed, but there is little understanding of the cell source-dependent trans-differentiation potential of MSCs into corneal epithelial cells. In the present study, we induced trans-differentiation of MSCs derived from umbilical cord matrix (UCM-MSCs) and from dental tissue (D-MSCs), and we comparatively evaluated the in vitro trans-differentiation properties of both MSCs into corneal epithelial-like cells. Specific cell surface markers of MSC (CD44, CD73, CD90, and CD105) were detected in both UCM-MSCs and D-MSCs, but MHCII and CD119 were significantly lower (P < 0.05) in UCM-MSCs than in D-MSCs. In UCM-MSCs, not only expression levels of Oct3/4 and Nanog but also proliferation ability were significantly higher (P < 0.05) than in D-MSCs. In vitro differentiation abilities into adipocytes and osteocytes were confirmed for both MSCs. UCM-MSCs and D-MSCs were successfully trans-differentiated into corneal epithelial cells, and expression of lineage-specific markers (Cytokeratin-3, -8, and -12) were confirmed in both MSCs using immunofluorescence staining and qRT-PCR analysis. In particular, the differentiation capacity of UCM-MSCs into corneal epithelial cells was significantly higher (P < 0.05) than that of D-MSCs. In conclusion, UCM-MSCs have higher differentiation potential into corneal epithelial-like cells and have lower expression of CD119 and MHC class II than D-MSCs, which makes them a better source for the treatment of corneal opacity.