• Journal of Plant Biotechnology
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• v.35 no.4
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• pp.275-280
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• 2008

One of the limitation for Agrobacterium-mediated transformation via organogenesis from cotyledon explants routinely in cucumber is the production of chimeric plants. To overcome the limitation, Agrobacterium-mediated transformation system via somatic embryogenesis from hypocotyl explants of cucumber (c.v., Eunsung) on the selection medium with paromomycin as antibiotics was developed. The hypocotyl explants were inoculated with Agrobacterium tumefaciens strain EHA101 carrying binary vector pPTN290; then were subsequently cultured on the following media: co-cultivation medium for 2 days, selection medium for $5{\times}14$ days, and regeneration medium. The T-DNA of the vector (pPTN290) carried two cassettes, Ubi promoter-gus gene as reporter and 35S promoter-nptll gene conferring resistance to paromomycin as selectable agent. The confirmation of stable transformation and the efficiency of transformation was based on the resistance to paromomycin indicated by the growth of putative transgenic calli on selection medium amended with 100mg/L paromomycin, and GUS gene expression. Forty eight clones (5.2%) with GUS gene expressed of 56 callus clones with resistance to paromomycin were independently obtained from 928 explants inoculated. Of 48 clones, transgenic plants were only regenerated from 5 clones (0.5%) at low frequency. The histochemical GUS assay in the transgenic seeds ($T_1$) also revealed that the gus gene was successfully integrated and segregated into each genome of transgenic cucumber.

• Journal of Plant Biotechnology
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• v.33 no.1
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• pp.27-32
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• 2006

To clarify the roles of bromelain in plants, we isolated BL1 gene encoding bromelain from pineapple stem tissues and sequenced. The full length cDNA is 933 bp and encodes a polypeptide of 311 amino acid residues. The cDNA is most similar 94% at the amino acid level to bromelain previously isolated from pineapple (BAA21929). Explants of Lactuca sativa were co-cultivated with Agrobacterium tume-faciences LBA 4404 strains containing nptII and BL1 gene for transformation. Through initial selection of regenerated explants by culturing on a kanamycin and carbenicillin containing MS medium, multiple shoots were obtained after 2 months of culture. For a complementary step of selection, putative transgenic shoots were transferred to 1/2 Ms basal medium supplemented with 100 mg/L kanamycin and 500 mg/L carbenicillin. The selected shoots were obtained T1 generation seeds with emasculation, and tested with PCR analysis using 35S promoter and BL1 specific primers whether BL1 gene was introduced to genome of the plants. These results confirmed that produced the specific PCR bands in the putative transgenic lines. Additionally the Northern blot and endo protease activity showed that transcripts of BL1 gene were detected in transgenic lines. Theses results suggest that BL1 gene be successfully integrated and transcripted in the transgenic lettuce plants.

• Journal of Plant Biotechnology
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• v.32 no.4
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• pp.257-262
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• 2005

Agrobacterium tumefaciens-mediated cotyledonary-node explants transformation was used to produce transgenic melon. Cotyledonary-node explants of melon (Cucumis melo L. cv. Super VIP) were co-cultivated with Agrobacterium strains (LBA4404, GV3101, EHA101) containing the binary vector (pPTN289) carrying with CaMV 35S promoter-gus gene as reporter gene and NOS promoter-bar gene conferring resistance to glufosinate (herbicide Basta) as selective agent, and the binary vector (pPTN290) carrying with Ubiquitin promoter-GUS gene and NOS promoter-nptll gene conferring resistance to paromomycin as selective agent, respectively. The maximum transformation efficiency (0.12%) was only obtained from the cotyledonary-node explants co-cultivated with EHA101 strain (pPTN289) on selection medium with 5 mg/L glufosinate and not produced a transgenic melon from the cotyledon or cotyledonary-node co-cultivated with other strains. Finally, five plants transformed showed the resistance in glufosinate antibiotic and the GUS positive response in leaf ($T_0$), flower ($T_0$), seeds ($T_1$) and plantlet ($T_1$). Southern blot analysis revealed that the gus gene integrated into each genome of transgenic melon.

• Korean Journal of Plant Tissue Culture
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• v.26 no.4
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• pp.281-287
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• 1999

Codonopsis lanceolata ("Deoduck" in Korea) is a perennial herb, and belongs to family, Campanulaceae. Its taproot is used a good source of a wild vegetable as well as an herbaceous medicine. In this study, to develop a bialaphos-resistant transgenic Codonopsis, seed germination mechanism and somatic embryogenesis of the plant were investigated, and Agrobacterium-mediated transformation with bar gene encoding phosphinothricin acetyltransferase (PAT) was performed. Attempt were made to regenerate plant from cells via somatic embryogenesis. When the cotyledons, nodes and leaf disks were cultured on MS medium containing 2,4-D and zeatin, embryogenic calli were induced. Upon transferring the somatic embryos to N6 solid medium without plant growth regulators, they developed into plantlets under continuous illumination. All plants were dead on MS basal medium containing 10 mg/L phosphinothricin (PPT) and Basta, respectively. The explants did not produce calli in the medium containing 200 mg/L kanamycin. The explants were cocultured with Agrobacterium tumefaciens for 2 days, and transformants were selected in MS basal medium containing 1.0 mg/L 2,4-D, 100 mg/L kanamycin and 500 mg/L carbenicillin. After the selection, embryogenic calli were induced and then somatic embryos were produced by subsequent subculturing. The somatic embryos were germiated on N6 basal medium containing 200 mg/L kanamycin and 500 mg/L carbenicillin. PCR analysis showed that nptII and bar genes were introduced in the Deoduck transformants. After the confirmation of bar gene expression in RNA and protein level, the transgenic Deoduck will be used to study the genetics of filial generation with the herbicide control gene, bar.gene, bar.

• Journal of Plant Biotechnology
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• v.35 no.1
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• pp.55-61
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• 2008

Previous studies on genetic transformation of chrysanthemum using cold regulated gene (BN115) have been conducted and the PCR and Real-Time PCR based method to determine the presence of the transferred cold regulated gene in the chrysanthemum was established. To check whether over-expression of BN115 gene in transgenic chrysanthemum will enhance their tolerance to cold stress, the transgenic chrysanthemum were grown under low temperature condition and several cold signalling including growth characteristics, stoma size and shape, SPAD value and ion leakage test were investigated. The transgenic chrysanthemum in the low temperature growth chamber grow much faster in term of the height, number and size of the leaves than those of wild-type plants and damage of transgenic plant caused by the low temperature was much less than that of wild-type plants. The stoma type and size of transgenic plant leaves grown at $5^{\circ}C$ were much similar to of wild-type plant cultured on $25^{\circ}C$ It has been found that SPAD value of transgenic plants was much higher than those of wild-type, but the EC density being lower under low temperature condition.

• Korean Journal of Plant Tissue Culture
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• v.26 no.3
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• pp.163-169
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• 1999

This study was conducted to produce herbicide resistant plants by transferring phosphinothricin acetyltransferase (PAT) gene into Populus alba $\times$ Populus glandulosa No .3 using Agrobacterium tumefaciens MP 90/PAT. Leaf segments from in vitro grown shoots of hybrid poplar No. 3 were soaked in a AB medium containing Agrobacterium tumefaciens MP 90/PAT for 10 min and cocultivated for 2 days on MS medium containing 1.0 mg/L 2,4-D and 0.2mg/L kinetin (CIM). Putative transformed calli could be selected after cocultivation of leaf segments on CIM supplemented with 50mg/L kanamycin and 500mg/L cefotaxime for 3 weeks. The selected calli were cultured on CIM supplemented with 50 mg/L kanamycin and 500 mg/L cefotaxime for 5~8 weeks before transfer to WPM containing 1.0mg/L zeatin, 0.1mg/L BAP, 50 mg/L kanamycin and 500mg/L cefotaxime for shoot regeneration. Shoots were regenerated from the callus after 4 week cultivation, and the regenerants were grown on the same medium for 7~l0 weeks. The plants rooted on 1/2 WPM containing 0.2 mg/L IBA and 50 mg/L kanamycin. To confirm the gene insertion into plants, GUS activity was detected by histochemical assay in the transformed plants. Finally, the presence of both NPT II and PAT genes from the transgenic plants were confirmed by PCR amplification with the gene specific primers and subsequent PCR-Southern blot with DIG-labeled PAT gene probe. After acclimatization in pots for 4 weeks, the plants were sprayed by 3 mL/L of Basta to test resistance to the herbicide. The transgenic plants remained green, whereas all the control plants died after one week.

• Journal of Plant Biotechnology
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• v.34 no.4
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• pp.375-380
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• 2007

This study was conducted to develop herbicide-resistance domestic maize plants by introducing the CP4 5-enol-pyruvylshikimate-3-phosphate synthase (CP4 EPSPS) gene using Agrobacterium tumefaciens-mediated immature embryo transformation. Immature embryos of five genotypes (HW1, KL103, HW3, HW4, HW7) were co-cultivated with strains Agrobacterium tumefaciens (strain C58C1) containing the binary vector (pCAMBIA2300) carrying Ubiquitin promoter-CP4 EPSPS gene and Cauliflower mosaic virus 35S (CaMV35S) promoter-nptll gene conferring resistance to paromomycin as a selective agent. The presence and expression of CP4 EPSPS transgene were confirmed by PCR, RT-PCR and Northern blot analysis, respectively. Also, the resistance to glyphosate in the transgenic maize ($T_1$) was analyzed by shikimate accumulation assay. The frequency (%) of paromomycin-resistance callus was 0.37, 0.03, 2.20, 2.37, and 0.81% in pure lines HW1, KL103, HW3, HW4 and HW7, respectively. EPSP transgene sequences were amplified in putative transgenic plants that regenerated from paromomycin-resistance calli of two inbred lines (HW3, HW4). Of them, RT-PCR and Northern blot analyses revealed that the transgene was only expressed in two transgenic events (M266, M104) of HW4 inbred line, and a mild glyphosate resistance of transgenic event (M266) was confirmed by the lower shikimate accumulation in leaf segments. These results demonstrate that transgenic maize with herbicide-resistance traits in Korean genotype can be genetically obtained.

• Korean Journal of Plant Tissue Culture
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• v.25 no.3
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• pp.207-217
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• 1998

Total of 78 strains were characterized based on the morphological characteristics of colonies isolated on Schroth, and New & Kerr's media for selection of hypervirulent wild-type Agrobacterium spp from galls, hairy root-like process and soil of Populus, Malus, Salix and Diopyros in Korea. Among them, 48 strains were able to induce tumors in carrot disc. Hypervirulent A. tumefaciens SP101 and SM042 were identified as biotype 1 and biotype 2, respectively, These strains formed fast growing, larger tumors as compared to those induced by other strains. The binary vector pGA643 with kanamycin resistant gene was mobilized from E. coli MC100 into A. tumefaciens strain SM042 isolated from soil, and/or disarmed vector PC2760 using a triparental mating method with E. coli HB101/pRK2013, and transconjugants, A. tumefaciens SM643 and PC643 were obtained in minimal media containing kanamycin and tetracycline. Tobacco tissues were cocultivated with conjugant Agrobacterium and then transferred to selective medium with 2,4-D and kanamycin to induce the transformants. Calli were formed more efficiently in cocultivation with A. tumefaciens SM643 than that with A. tumefaciens PC643. Most of calli transformed with A. tumefaciens PC643 were friable and regenerated into normal plantlets, while the calli transformed with A. tumefaciens SM643 were compact, hard, and mixed with friable calli. The friable calli formed normal shoots, while compact calli did not form shoots but only grew to typical compact tumor calli. When the shoots formed directly from tobacco stems without callus induction after transformation by A. tumefaciens SM643 with wild-type Ti-plasmid, normal transformed plants can be induced without using disarmed Ti-plasmid.

• Journal of Plant Biotechnology
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• v.35 no.4
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• pp.299-306
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• 2008

A modified method of Agrobacterium-mediated perilla transformation was developed using two selection markers of an antibiotics (either hpt or nptll) and an herbicidal (bar) gene. Perilla hypocotyl explants were cocultured with Agrobacterium tumefaciens EHA 105 strain harboring plasmid vector (either pMOG6-Bar or pCK-Bar) for three days, respectively. Primary shoots were selected with antibiotics of hygromycin (15 mg/L) or kanamycin (125 mg/L) and regenerated shoots were further selected with herbicide phosphinothricin (ppt,1.2 mg/L) to obtain authentic transformants. Roots were induced for the regenerated shoots on the MS medium without hormone and 80 putative transgenic plants were obtained. Transgene integration into perilla genome was confirmed by Southern blot and their expression was analyzed by Northern blot. T1 perilla seeds drived from To plants were tested 0.3% basta spray for identification of stable gene delivery to next generation.

• Journal of Plant Biotechnology
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• v.34 no.1
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• pp.37-45
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• 2007

The genetic transformation of watermelon by Agrobacterium has been known very difficult and a few successful cases have been reported by obtaining the direct shoot formation. However, since this direct shoot formation is not guaranteed the stable transformation, the stable transformation with reproducibility is required by a different approach such as a callus induced manner. The best conditions for inducing the callus from cotyledon and root explants of watermelon were 2 mg/L zeatin + 0.1 mg/L IAA and 2 mg/L BA + 0.1 mg/L 2,4-D, respectively. The GFP expression in the callus was identified and monitored through fluorescent microscopy after transformation with pmGFP5-ER vector. Paromomycin rather than kanamycin was used for selecting the nptll gene expression because it was more effective to select the watermelon explants. Four different callus types were observed and the solid green callus showed stronger GFP expression. The highest frequency of GFP expression in the callus developed from cotyledon was 9.0% (WM8 inbred line), while the highest frequency from root was 8.3% (WM6 inbred line). The WMV-CP was transformed using the method of GFP transformation and the genetic transformation of WMV-CP was confirmed by PCR and Southern blot analysis. Here we present a system for callus induction of watermelon explant and the callus induced method would facilitate the establishment of stable watermelon transformation.