• Reproductive and Developmental Biology
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• v.36 no.1
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• pp.49-53
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• 2012

The present study was conducted to examine the generation of reactive oxygen species (ROS) during micromanipulation procedures in bovine somatic cell nuclear transfer (SCNT) embryos. Bovine enucleated oocytes were electrofused with donor cells, activated by a combination of Ca-ionophore and 6-dimethylaminopurine culture. Oocytes and embryos were stained in dichlorodihydrofluorescein diacetate or 3'-(p-hydroxyphenyl) fluorescein dye and the $H_2O_2$ or $^.OH$ radical levels were measured. $In$ $vitro$ fertilization (IVF) was performed for controls. The samples were examined with a fluorescent microscope, and fluorescence intensity was analyzed in each oocyte and embryo. The $H_2O_2$ and $^.OH$ radical levels of reconstituted oocytes were increased during manipulation (37.2~49.7 and 51.0~55.2 pixels, respectively) as compared to those of mature oocytes ($p$<0.05). During early $In$ $vitro$ culture, the ROS levels of SCNT embryos were significantly higher than those of IVF embryos ($p$<0.05). These results suggest that the cellular stress during micromanipulation procedures can generate the ROS in bovine SCNT embryos.

• Korean Journal of Plant Tissue Culture
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• v.25 no.1
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• pp.1-5
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• 1998

This study describes the effect of the growth regulators such as 2,L-D and BA, on the structural abnormalities of somatic embryos derived from leaf explants of Angelica gigas Nakai, Also, the relationship between the cotyledon number of a somatic embryo and its germinability is explored. Embryogenic calli were selected from calli formed on explants cultured on MS solid basal medium supplemented with 0.5mg/L 2,4-D, 1mg/L 2,4-D, 1mg/L plus 0.1mg/L BA, and 1 mg/L 2,4-D plus 0.5mg/L BA. Cotyledonary abnormalities were observed in somatic embryos which were developed from embryogenic calli cultured on MS medium containing 1mg/L 2,4-D for 8 weeks and then subcultured on 2,4-D free MS medium for 3 weeks. The frequency of abnormalities was as follows: 22.8% one cotyledon, 42.5% two cotyledons, 16.8% three cotyledons, 7.8% four cotyledons, 1.8% five cotyledons, and 8.2% jar shaped cotyledon. In addition, ABA treatment indicated an improvement of the somatic embryo with normal cotyledon (65.3%). ABA was important role to the high production of normal somatic embryos. Two cotyledon embryos showed germinability 77.8%. However the germinability of somatic embryos with anomalous cotyledons was prominently low: One cotyledon, 62.5%; three cotyledons, 43.3%; four cotyledons, 60%; five cotyledons, 50% and jar shaped cotyledon, zero%. Thus, germinability was essentially, inversely proportional to cotyledon number.

• Journal of Plant Biotechnology
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• v.47 no.2
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• pp.179-184
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• 2020

Embryogenic calli from Codonopsis lanceolata L. were cultured in MS liquid media and supplemented with various concentrations of primary carbon sources to study the effects of carbohydrates and osmoticum on somatic embryogenesis and somatic embryo morphology. Sucrose, glucose, and a combination of 3% sucrose and various concentrations of sorbitol or mannitol as osmoticum were used as carbon supplements. The maximum number of somatic embryos per flask was greater in media exclusively supplemented with 3% sucrose (128.29) than exclusively glucose-supplemented media (47.67) and either supplement combination of 3% sucrose and osmoticum (95.67 with mannitol and 114.00 with sorbitol). The number of somatic embryos gradually decreased in media with increasing concentrations of combined osmoticum supplement. Decreases also occurred in the highest concentrations of sucrose- and glucose-supplemented media. The total frequency of somatic embryos with two cotyledons was slightly higher in medium with 3% + mannitol (24.09%) compared with exclusively sucrose (21.52%), glucose (21.22%), or 3% sucrose + sorbitol (22.13%). As concentrations of sucrose and glucose increased, the occurrence of two cotyledons and trumpet cotyledons gradually decreased and the occurrence of polycotyledon and globular stage embryos increased. Furthermore, as concentrations of 3% sucrose and osmoticum increased, the occurrence of trumpet cotyledon and globular stage embryos increased and the occurrence of polycotyledon gradually decreased. These results demonstrated that the somatic embryogenesis and occurrence of cotyledon morphology were influenced by the concentration of carbohydrates and combinations of 3% sucrose and osmoticum supplements.

• Korean Journal of Plant Tissue Culture
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• v.21 no.1
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• pp.1-6
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• 1994

Cotyledonary and hypocotyl explants of melon seedlings were cultured on Murashige and Skoog's (MS) medium supplemented with various concentrations of 2,4-Dichlorophenoxyacetic acid (2,4-D) and benzyladenine (B.A).Up to 22% of cotyledonary explants and 7%, of hypocotyl explants, respectively: Produced somatic embryos through intervening two types of calli: bright yellow compact (BYC) callus and pale-yellow compact (PYC) callus. BYC callus was capable of producing somatic embryos at initial culture, but it became necrotic as subrulhues proceeded. In contrast UC callus was incapable of producing somatic embryos during initial culture (first 6 weeks), but it became bright-yellow friable (BYF) callus with forming a few globular embryos after 2 months of subculture, indicating that the callus turned embryogenic. The embryogenic capacity of BYF maintained for over one year when the callus was sucultured at 4-week interval. Upon transfer onto MS basal medium the callus gave rise to numerous somatic embryos and subsequently converted to plantlets. Plantlets were transplanted to potting soil and grown to maturity in the phyotron.

• Plant Biotechnology Reports
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• v.3 no.3
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• pp.205-211
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• 2009

An efficient in vitro regeneration protocol for moth bean [Vigna aconitifolia (Jacq.) Marechal] via somatic embryogenesis has been developed. Embryogenic callus cultures were established from the cotyledonary node as explant on semi-solid Murashige and Skoog (MS) medium supplemented with $0.75mg\;1^{-1}$ 2,4-dichlorophenoxyacetic acid (2,4-D) and $1.5mg\;1^{-1}$ 6-benzylaminopurine (BA) and with various additives ($50mg\;1^{-1}$ ascorbic acid and $25mg\;1^{-1}$ each of adenine sulphate, citric acid and $_L-arginine$). Numerous somatic embryos differentiated on MS basal nutrient medium supplemented with $0.25mg\;1^{-1}$ 2,4-D and $0.5mg\;1^{-1}$ of kinetin (Kin). Sustained cell division resulted in the formation of cell aggregates, which progressed to the globular- and heart-shaped somatic embryos and then, if they differentiated properly, to the torpedo shape and cotyledonary stages. The transfer of embryos onto fresh MS basal medium containing $0.2mg\;1^{-1}$ BA and $2.0mg\;1^{-1}$ gibberellic acid enabled the embryos to achieve complete maturation and germination. More than 80% of somatic embryos were converted into true-to-type fertile plants. In vitro-regenerated plantlets with well-developed roots were successfully hardened in a greenhouse and established in soil.

• Journal of Crop Science and Biotechnology
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• v.11 no.2
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• pp.119-126
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• 2008

Since somatic embryogenesis combined with ethylmethane sulfonate(EMS) treatments is the most efficient technique for mutagenesis, the embryogenic capacity of four soybean cultivars was evaluated at different EMS concentrations, treatment times, and preculture durations. Two to 4 mm long immature cotyledons were placed in induction medium after EMS treatment, and the numbers of somatic embryos formed per explant were counted four weeks after culture initiation. We observed genotypic differences in the efficiency of somatic embryogenesis from immature embryos among four cultivars treated with different concentrations of EMS for six hours. Cultivars, Sinpaldalkong 2 and Jack, displayed highly efficient somatic embryogenesis regardless of EMS concentration, whereas very low efficiency or no survival was observed in Jinju 1 and Iksannamulkong cultivars. Preculture duration did not influence the efficiency of somatic embryogenesis. Because Sinpaldalkong 2 exhibited the best somatic embryogenesis, much higher concentrations of EMS were used to test somatic embryo formation under different periods of time in this cultivar. Three and six hour treatments with both 1 and 2 mM EMS yielded higher embryo formation than longer periods of time. Increasing the time with embryos in 2 mM EMS caused a reduction in somatic embryogenesis in Sinpaldalkong 2, but many chlorophyll-deficient soybean variants were identified in the $M_1R_0$ and $M_2R_1$ generations. In addition to Jack, Sinpaldalkong 2 is a good genotype for plant regeneration from EMS-treated immature embryo cultures.

• Journal of Crop Science and Biotechnology
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• v.11 no.2
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• pp.107-110
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• 2008

A high-efficiency, reproducible somatic embryogenesis system for strawberry cultivar Chandler was developed. Thirty-one somatic embryos per explant(max no.) were recorded in leaf discs which were cultured on medium containing MS salts+$B_5$ vitamins+2% glucose+4.0 mg $1^{-1}$TDZ(Thidiazuron) and incubated at $10{\pm}1^{\circ}C$ under darkness for one week followed by three weeks under 16-h photoperiod. The scanning electron microscopic(SEM) ontogeny revealed the normal development of somatic embryos from globular to heart-shaped and dissection microscopy from torpedo-shaped to cotyledonary-stage embryos. The maximum germination percentage of 48% could be obtained on MS medium containing kinetin(1.0 mg $1^{-1}$) and the maximum survival percentage(79%) of plantlets after four weeks was found to be in the mixture of vermiculite, peatmoss, and soilrite(1:1:1).

• Korean Journal of Plant Tissue Culture
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• v.26 no.3
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• pp.143-148
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• 1999

This study was conducted to elucidate the effects of ascorbic acid and dehydroascorbic acid on somatic embryogenesis from the cultured cells of carrot. Ascorbic acid in culture medium merely stimulated the proliferation of non-embryogenic cells but dehydroascorbic acid in medium induced embryogenic cells from non-embryogenic cells accompanying the inhibition of cell proliferation. Ascorbic acid in medium inhibited somatic embryogenesis from embryogenic cells while dehydroascorbic acid in medium enhanced somatic embryogenesis from the cells as well as non-embryogenic cells. This enhancement was limited to globular embryos and the maturation to cotyledonary embryos was inhibited by dehydroascorbic acid treatment. From the above results it is suggested that carrot callus cultures on medium containing dehydroascorbic acid could quickly induce embryogenic cells. In addition after brief culture of embryogenic cells on development medium containing dehydroascorbic there by acid the subculture of the cells to MS basal medium resulted in the high frequency production of somatic embryos.

• Journal of Plant Biotechnology
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• v.30 no.4
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• pp.359-363
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• 2003

Mass propagation of tulip tree (Liriodendron tulipifera L) via somatic embryogenesis was successfully achieved with immature samaras collected from adult trees. Embryogenic tissues were induced by culturing them samaras on 1/2 LM medium (Litvay's) containing 2,4-D and BA. Somatic embryos developed from the embryogenic tissues and germinated to normal plants (emblings) upon transfer onto the same medium containing either AgNO$_3$ or activated charcoal. So far, several factors appeared to influence both the induction of embryogenic tissues and germination of the embryos into plants. These include the collection time of samaras for the induction of embryogenic tissue, sucrose level in the culture medium, the level of both AgNO$_3$ and activated charcoal, and plating density of somatic embryos on germination medium for maturation and germination of somatic embryos into plantlets.

• Korean Journal of Medicinal Crop Science
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• v.5 no.4
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• pp.289-293
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• 1997

The effects of plant regulators on direct somatic embryogenesis in liquid culture of Rehmannia glutinosa were investigated and the proper explant for direct somatic embryo formation was studied. Direct somatic embryos were induced from leaf segments culture in the MS liquid medium containing 0.5 mg/l of both IAA and NAA, while IBA of 1.0 mg/l was required for the same effect. Many somatic embryos were directly formed at the concentration of 2.0 mg/l cytokinin such as BA, kinetin and zeatin, but somatic embryogenesis was relatively poor at above or below this level. Relatively more somatic embryos were induced in the combination of 1.0mg/l IAA and 2.0mg/l zeatin. Formation of somatic embryos begun after 6 weeks on stem segments, while 7 weeks both on petiole and leaf. However, overall production of somatic embryos after 8 weeks was higher in leaf segment than that of stem segment.