• Journal of Plant Biotechnology
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• v.6 no.4
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• pp.229-233
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• 2004

Partial desiccation of embryogenic calli cultures or somatic embryos leads to different physiological changes and maturation of somatic embryos, leading to improved plant regeneration. Embryogenic calli was induced from immature inflorescence segments and young leaf rolls of sugarcane (Saccharum officinarum hybrids CoC-671) on Murashige and Skoog's basal medium enriched with different concentrations of 2,4-D ($1-4\;\cal{mg/l}$), L-glutamine ($100\cal{mg/l}$), malt extract ($100\cal{mg/l}$), casein hydrolysate ($1000\;\cal{mg/l}$) and coconut milk ($5\%$) and solidified with $0.2\%$ gel rite. The embryogenic calli were subjected to desiccation for 1-8 h. Desiccation of the calli for 6-7 h resulted in enhancement of plant regeneration frequency ($83-96\%$) as compared to control ($12\%$). Plantlets exhibited vigorous growth to maturity in the greenhouse. Partial desiccation of embryogenic calli offers as a simple method for improving plant regeneration frequency in sugarcane.

• Korean Journal of Plant Resources
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• v.24 no.3
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• pp.280-285
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• 2011

In this study, we established a novel somatic embryogenesis and plant regeneration system through cell suspension culture of white dandelion (Taraxacum coreanum NAKAI.). Embryogenic calli could be initiated from leaf and root explants of sterile seedlings on solid Murashige and Skoog (MS) medium supplemented with 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) after 3-week cultures. To proliferate embryogenic calli rapidly, cell suspension culture was performed with transferred to liquid MS medium with various combinations of plant growth regulators (PGRs) including 2,4-D, ${\alpha}$-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA), $N^6$-benzylamino purine (BAP), thidiazuron (TDZ), and kinetin. During suspension cultures, embryogenic calli not only greatly proliferated, but shoot organogenesis also simultaneously occurred from the surface of somatic embryos. Among them, TDZ at lower concentration, 0.1 mg/L produced the highest efficiency of somatic embryo formation and shoot organogenesis. Rooting of embryogenic calli with adventitious shoots was done on solid MS medium containing 0.1 mg/L NAA and 0.3% activated carbon. Nearly 80% of embryogenic calli with shoot organogenesis could be rooted normal. Well-rooted plantlets were transferred into pots under a greenhouse condition, and plants derived from this system appeared phenotypically normal.

• Korean Journal of Plant Tissue Culture
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• v.28 no.5
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• pp.277-281
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• 2001

Embryogenic callus induction and plant regeneration methods were developed for native Kentucky bluegrass (Poa pratenes L.) ecotypes. Mature caryopses and immature inflorescences (20 mm in length) of 4 native ecotypes and 5 foreign cultivars were plated on MS medium (30 g/L sucrose, 3 g/L Phytagel) supplemented with 1 mg/L 2,4-D, and cultured in the dark at 24$^{\circ}C$. Most explants formed calli, but more embryogenic calli were induced from the explants of immature inflorescences than caryopses which produced mostly non-embryogenic rooty calli. In P77 ecotypes, immature inflorescence explants formed embryogenic calli with the rate of 62~95%, and those of field-grown plants were more efficient than greenhouse-grown ones in embryogenic callus induction. Plantlets were regenerated from the embryogenic calli when they were transferred to hormone-free MS medium, and grew to maturity without morphological variations in greenhouse.

• Plant Biotechnology Reports
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• v.3 no.4
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• pp.277-283
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• 2009

One of the limitations to conducting maize Agrobacterium-mediated transformation using explants of immature zygotic embryos routinely is the availability of the explants. To produce immature embryos routinely and continuously requires a well-equipped greenhouse and laborious artificial pollination. To overcome this limitation, an Agrobacterium-mediated transformation system using explants of type II embryogenic calli was developed. Once the type II embryogenic calli are produced, they can be subcultured and/or proliferated conveniently. The objectives of this study were to demonstrate a stable Agrobacterium-mediated transformation of maize using explants of type II embryonic calli and to evaluate the efficiency of the protocol in order to develop herbicide-resistant maize. The type II embryogenic calli were inoculated with Agrobacterium tumefaciens strain C58C1 carrying binary vector pTF102, and then were subsequently cultured on the following media: co-cultivation medium for 1 day, delay medium for 7 days, selection medium for $4{\times}14$ days, regeneration medium, and finally on germination medium. The T-DNA of the vector carried two cassettes (Ubi promoter-EPSPs ORF-nos and 35S promoter-bar ORF-nos). The EPSPs conferred resistance to glyphosate and bar conferred resistance to phosphinothricin. The confirmation of stable transformation and the efficiency of transformation was based on the resistance to phosphinothricin indicated by the growth of putative transgenic calli on selection medium amended with $4mg\;1^{-1}$ phosphinothricin, northern blot analysis of bar gene, and leaf painting assay for detection of bar gene-based herbicide resistance. Northern blot analysis and leaf painting assay confirmed the expression of bar transgenes in the $R_1$ generation. The average transformation efficiency was 0.60%. Based on northern blot analysis and leaf painting assay, line 31 was selected as an elite line of maize resistant to herbicide.

• Korean Journal of Plant Tissue Culture
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• v.22 no.5
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• pp.283-290
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• 1995

This experiment was canted out to obtain embryogenic callus and to understand developmental mechanism of somatic embryogenesis in Oenanthe javanica ($B_{L.}$) DC. experiments included the examination of explant source and media for embryogenic callus production and the observation of developmental pattern of embryogenic cells and non-embryogenic cells. Embryogenic calli were formed on zygotic pro-embryos together with their endosperms when they were cultured on Ms media containing 1.0mg/L 2,4-D. Embryogenic calli were also formed on the intact surface in vitro grown stem or petiole segmentsafrer 6-8 weeks of culture, whereas non-embryogenic calli were formed on cut surfaces of the stem and petiole after 2 weeks of culture. Non-embryogenic calli were rhizogenic in suspension and solid media culture.

• Korean Journal of Plant Tissue Culture
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• v.26 no.4
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• pp.289-291
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• 1999

Hypocotyl explants from 7 days old seedlings of one $F_1$ hybrid cultivar and two pure lines of cucumber formed embryogenic calli at frequencies of up to 8% when cultured on Murashige and Skoog medium (MS) supplemented with 1 mg/L 2,4-D for 3 weeks. Embryogenic calli gave rise to somatic embryos. When slices of somatic embryos were cultured on the same medium for 4 weeks, they formed embryogenic calli. Embryogenic cell suspension cultures were established with embryogenic calli in MS liquid medium with 1 mg/L 2,4-D. Embryogenic potential of cell suspension cultures was maintained by subculturing every seven days. When the level of 2,4-D in the medium was lowered to 0.2 mg/L by diluting with liquid MS basal medium, embryogenic cell suspension cultures underwent development into numerous somatic embryos. When plated onto MS basal medium, over 95% of somatic embryos developed into plantlets. Plantlets were transplanted to potting soil and grown to maturity.

• Journal of Plant Biotechnology
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• v.7 no.4
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• pp.211-218
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• 2005

Genetic transformation was carried out by using biolistic gun method. The hypocotyl derived embryogenic calli (explants) of cotton (Gossypium hirsutum L.) cv. Cocker-312 were transformed with a recombinant pGreen II plasmid, in which both, bar (selection marker) and GUS (${\beta}$-glucuronidase) reporter genes were incorporated. Explants were arranged on osmoticum-containing medium (0.5M mannitol) 4 hours prior to and 16 hours after bombardment that was resulted into an increase about >80% for GUS stable expression. 3 days after bombardment, GUS assay was performed, which exhibited, $18.36{\pm}1.00$ calli showed blue spots. The transformed embryogenic calli were cultured on selection medium (@ 6 mg/L basta) for 3 months. The putative transgenic plants were developed via selective somatic embryogenesis (@1.50 mg/L basta); maximum $27.58{\pm}1.25$ somatic embryos were obtained while $17.47{\pm}1.00$ embryos developed into plantlets (@ 0.75mg/L basta). In five independent experiments, up to 7.24% transformation efficiency was recorded. The presence of the transgenes was analyzed by using PCR and southern hybridization analysis. The transgenic plants were developed with in 6-7 months, but mostly transformants were abnormal in morphology.

• Journal of Plant Biotechnology
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• v.31 no.4
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• pp.267-271
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• 2004

Transformed sweetpotato (Ipomoea batatas (L.) Lam. cv. Yulmi) plants were developed from embryogenic calli following Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105/pCAMBIA2301 harboring genes for intron $\beta$-glucuronidase (GUS) and kanamycin resistance. Transient expression of GUS gene was found to be higher when embryogenic calli were co-cultivated with Agrobacterium for 2 days. The co-cultured embryogenic calli transferred to selective MS medium containing 1mg/L 2,4-D, 100mg/L kanamycin, and 400mg/L claforan. These embryogenic calli were subcultured to the same selection medium at 4 weeks interval. Kanamycin-resistant calli transferred to hormone-free MS medium with kanamycin gave rise to somatic embryos and then converted into plantlets in the same medium. Southern blot analysis confirmed that the GUS gene was inserted into the genome of the sweetpotato plants. A histochemical assay revealed that the GUS gene was preferentially expressed in the leaf, petiole, and vascular tissue and tip of root.

• Korean Journal of Medicinal Crop Science
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• v.7 no.4
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• pp.275-281
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• 1999

Embryogenic calli of Codonopsis lanceolata were cultured on MS agar medium containing various concentrations of sucrose as a carbon source. Upon transfer to MS basal medium, somatic embryos of cotyledonary stage converted to plantlets. When sucrose was added with greater than 4%, the number of shoots and roots regenerated from somatic embryo increased. However, the growth of shoots and roots was retarded in agar medium with more than 2% sucrose, but promoted in medium with lower concentration of sucrose. Saponin contents of shoots regenerated from somatic embryos, embryogenic calli, non-embryogenic calli, and native roots were determined by HPLC. Saponin contents of native root was variable, depending on regenerant, embryogenic calli, and cotyledonary embryos. The saponin contents of regenerated roots in medium with high sucrose was similar to native roots. Saponins content based on cell differentiation to shoot and root was dramatically decreased. This results could be effectively controlled for the production of useful secondary metabolites.

• Korean Journal of Plant Tissue Culture
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• v.27 no.3
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• pp.213-217
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• 2000

To investigate the cucumber regeneration from embryogenic calli, shoot tips of aseptically-grown cucumber seedlings were used as explants for establishing tissue cultures. Growth and differentiation of callus were studied by using Murashige and Skoog's (MS) medium containing 0.5 to 2 mg/L 2,4-D. Plantlets were induced from shoot tip culture on the plant growth regulators-free MS medium. Non-embryogenic calli and viscous calli were induced on the medium supplemented with 0.5 to 2 mg/L 2,4-D, but embryogenic callus was not induced on the same medium. Segments (ca. 5∼10 mm) of aseptically-grown hypocotyl from five to seven days old seedlings after germination were placed on MS medium supplemented with 1 mg/L 2,4-D for 50 days. Embryogenic calli and embryoids were induced only from the seedlings grown in dark condition, and hypocotyl was placed on the media explanted in light condition. Foully-five point one percent of white fragile calli and 0.6% yellowish compact calli formed roots. Yellowish callus lines were investigated to have a considerably higher concentration of crude proteins than white callus lines. Plantlets derived from embryogenic calli or embryoids have been transferred to pots containing sterile vermiculite and perlite. Normal fruits were harvested from nutrient culture on aggregated hydroponics in the F-clean house.