• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.146.1-146
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• 2003

Transgenic Nicotiana benthmiana plants harboring and expressing coat protein (CP) gene of Zucchini green mottle mosaic virus (ZGMMV) were generated for both virus-resistant screening and complementation analysis of related viruses and environmental safety assessment (SA) of living modified organism (LMO) purposes. Transformation of leaf disc of N. benthamiana was performed using Agrobacterium-mediated method and the pZGCPPGA748 containing the ZGMMV CP and NPTII genes. Two kinds of transgenic homozygous groups, virus-resistant and -susceptible lines, were obtained by screening of challenging homologous virus for T1 generations. Complementation of CP-deficient related virus was analyzed using the susceptible line of ZGMMV. These two pathologically different lines can be useful for host-virus interactions and LMO environmental SA.

• Journal of Plant Biotechnology
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• v.37 no.3
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• pp.313-318
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• 2010

To develop edible vaccines for swine, the embryogenic calli (type II) derived from HiII genotype were inoculated with A. tumefaciens strain C58C1 containing the binary vector pMYV611, 613, 616, and V621, 622 and 623 respectively. Six of those vectors carry nptII gene which confers resistance to paromomycin and apxIIA gene producing ApxII toxin which is generated in various serum types of A. pleuropneumoniae as a target gene. The 4,120 callus clones for pMYV611, 5,959 callus clones for pMYV613, 7,581 callus clones for pMYV616, 52,329 callus clones for V621, 48,948 callus clones for V622, and 56,188 callus clones for V623 were inoculated. The frequency of positive response clone was confirmed into range of 2.3% - 4.4% for each vectors by NPTII ELISA kit assay, and the selected callus clones of them were finally 3 callus clones from pMYV611 (0.07%), 4 callus clones from pMYV613 (0.07%), 2 callus clones from pMYV616 (0.03%), 51 callus clones from V621 (0.1%), 72 callus clones from V622 (0.15%), and 102 callus clones from V623 (0.18%) respectively. From the selected callus clones of each binary vector, the integration of the apxIIA gene into maize genome was detected from 2 plants of pMYV613 and 2 plants of V623 by Southern blot analysis.

• Korean Journal of Plant Tissue Culture
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• v.22 no.6
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• pp.329-334
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• 1995

Transformation system of rolC gene, dwarf gene in Lycium chinenese Mill. established by using system. Pin-punctured leaves induced numerous adventious buds in abaxial side when cultured on 3/2 MS medium containing 2.0 mg/L zeatin. Survival rate and shoot regeneration frequency of leaf explants decreased as kanamycin sulfate level increased. Shoot buds were not regenerated on 3/2 MS medium containing 10 mg/L kanamycin sulfate and 2.0 mg/L zeaein. Of the level tested, 10 mg/L of kanamycin sulfate was optimum in selection of kanamycin sulfate resistant plant. Co-culture time of bacteria and leaf explants was affected at the frequency of shoot regeneration and survival of leaf explants. Leaf explants co-cultivated during above 48hr severely decreased survival rate and shooting rate. Best result on survival rate and shooting rate were obtained when exposed for 24 h. 80 explants of 105 leaf explants survived on 3/2 MS medium containing 2.0 mg/L zeatin and 10 mg/L kanamycin sulfate, and 15 shoots was regenerated on the same medium. To select kanamycin sulfate resistant plant, regenerate as cultured on 3/2 MS medium containing 10 mg/L kanamycin sulfate, and obtained 5 kanamycin resistant plants. Southern blot analysis conformed that the rolC gene was incorporated into the genomic DNA of kanamycin resistant plants.

• Korean Journal of Plant Tissue Culture
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• v.28 no.5
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• pp.243-248
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• 2001

This study was peformed to find out the optimal conditions for the selection of transformed cells using already established transgenic plants. Several transgenic poplar (Populus alba$\times$P giandulosa) lines carrying npt II or hpt gene as a selectable marker were tested against kanamycin or hygromycin. Two culture explants, leaf discs and nodes, were compared regarding their sensitivity to the antibiotics. When leaf discs of untransformed control plants were cultured on callus inducing media in the presence of varying levels of kanamycin or hygromycin, only those cultured on the media containing lower than 50 mg/L kanamycin or 2 mg/L hygromycin formed callus. However, much higher concentration of kanamycin was needed to suppress the growth of axillary buds of untransformed plants. On the other hand, hygromycin at the concentration of 5 mg/L effectively suppressed shoot growth of untransformed plants. Root induction from untransformed plants could also be suppressed at the concentration of 50 mg/L kanamycin or 5 mg/L hygromycin. The transgenic plants showed resistance to 100 mg/L kanamycin or 50 mg/L hygromycin in the growth of callus, shoots, and roots. Hygromycin appeared to be more efficient in selecting untransformed cells than kanamycin.

• Korean Journal of Weed Science
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• v.17 no.4
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• pp.390-399
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• 1997

This experiment was conducted to introduce PAT (phosphinothricin acetyltransferase, non-selective herbicide bialaphos resistant gene) gene into potato (Solanum tuberosum. cv. Desiree). Optimal shoot regeneration from leaf discs and stem segments was obtained in MS medium supplemented with 0.1 mg/L IBA and 0.5 mg/L BA, and the frequency of shoot regeneration was 54% in left discs and 46% in stem segments. In this condition, leaf discs and stem segments of potato were co-cultivated with A. tumefaciens MP90 which contained binary vector with GUS: :NPTII gene and PAT gene. Transgenic shoots were regenerated from leaf and stem-derived calli on selection medium with 100mg/L kanamycin. The 100${\mu}M$ acetosyringone treatment during the co-cultivation highly enhanced(4 times than the control) the shoot regeneration on selection medium. When the putative transgenic plants were transferred to medium with 10mg/L basta, all of them were survived. After PCR. GUS test, and Southern blot analysis of the survived plant, we confirmed that the gene was stably integrated into the potato genome and expressed. After the transgenic plants were transplanted in soil, and the transgenic plants were sprayed with the herbicide basta (300ml/10a), the transgenic plants remained green but control plants were died.

• The Plant Pathology Journal
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• v.20 no.3
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• pp.206-211
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• 2004

Transgenic Nicotiana benthamiana plants harboring coat protein (CP) gene of Zucchini green mottle mosaic virus (ZGMMV) were generated for virus-resistant screening and complementation analysis of related viruses for environmental safety assessment (SA) of living modified organism (LMO) purposes. Transformation of leaf disc of N.benthamiana was performed by using Agrobacterium-mediated method and the pZGC-PPGA748 containing the ZGMMV CP and NPTII genes. Two kinds of transgenic homozygous groups, virus-resistant and virus-susceptible N.benthamiana lines, were obtained by screening of challenging homologous virus for Tl generations. These two pathologically different lines can be useful for host-virus interactions and LMO environmental SA.

• KSBB Journal
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• v.16 no.5
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• pp.480-485
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• 2001

Genetic transformation in dandelion(Taraxacum mongolicum Hand). was studied. We used for transformation by Agrobacterium tumefaciens strian LBA4404 harboring a binary vector pBI121 carrying the CaMV 35S promoter-GUS gene fusion used as a reporter gene and NOS promoter-NPTII gene as a positive selection marker. To obtain transformed plants, leaf explants of dandelion were cocultured with Agrobacterium tumefaciens LBA4404 for 10 mins, then transferred to MS medium containing 1 $\mu$M IAA, 1$\mu$M BA, 100$\mu$g/ML carbenicillin and 50 $\mu$g/ML kanarmycin sulfate. After two weeks of subculture of the explants, Kanamycin-resistant shoots were formed on explants survived. When subjected to GUS histochemical assay, all of the regenerants showed the GUS-positive responses. Plantlets were be be transformed to soil for further growth.

• Journal of Plant Biotechnology
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• v.30 no.2
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• pp.143-149
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• 2003

This experiment was carried out to confirm the stability of bar gene introduced into petunia plant through Agrobacerium-mediated transformation. Twenty-five transgenic plants T$_{0}$ plants, back cross (BC$_1$) populations to wild type and F$_1$plants between different T$_{0}$ plants were prepared, and polymerase chain reaction(PCR), PCR-Southern blot analysis, and field test with 0.1% Basta treatment were done. The results of PCR, PCR-Southern blot hybridization, and field test indicated that NPTII and bar gene introduced into the genome of petuina plants were stably transmitted to their progenies, and conferred the plants resistance to herbicide, Basta.sta.

• Journal of Plant Biotechnology
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• v.6 no.1
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• pp.15-19
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• 2004

The expression of a chimeric transgene (nos-npt II) has been examined in 9 years old transgenic poplars (Populus koreana x P. nigra) growing in a nursery. The expression of the gene in twenty six independentely transformed plants were examined by 1) enzyme (NPT II) assay, 2) RT-PCR, and 3) resistance to kanamycin. High NPT II activities in young leaves of all the transformed plants were found even without a selection pressure for antibiotics for 9 years. However, the activity varied with the positions of leaves in the stem in that young leaves showed higher activity than did mature tissues. When leaf segments were cultured in the presence of 150 mg/l kanamycin, only those from young leaves produced vigorously growing callus. However, as in the case of NPTII assay, the leaf segments from mature leaves did not form callus well on the media. RT-PCR with nptII specific primers also showed that amplification products were observed only when RNAs from young tissues were used. The total RNA gel showed that while RNA in young leaves are relatively stable and in a large quantity, those in old leaves were mostly degraded. All the above results suggest that the gene is transcriptionally active only in young tissue even though it is attached to a constituitive promoter. Therefore, the expression of foreign gene in poplar plants seemed to be affected by the metabolic state of the cells and thus vary greatly with the developmental stages and the age of tissue.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.10
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• pp.1390-1394
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• 2004

We have achieved efficient transformation system for forage-type tall fescue plants by Agrobacterium tumefaciens. Mature seed-derived embryogenic calli were infected and co-cultivated with each of three A. tumefaciens strains, all of which harbored a standard binary vector pIG121Hm encoding the neomycin phosphotransferase II (NPTII), hygromycin phosphotransferase (HPT) and intron-containing $\beta$-glucuronidase (intron-GUS) genes in the T-DNA region. Transformation efficiency was influenced by the A. tumefaciens strain, addition of the phenolic compound acetosyringone and duration of vacuum treatment. Of the three A. tumefaciens strains tested, EHA101/pIG121Hm was found to be most effective followed by GV3101/pIG121Hm and LBA4404/pIG121Hm for transient GUS expression after 3 days co-cultivation. Inclusion of 100 $\mu$M acetosyringone in both the inoculation and co-cultivation media lead to an improvement in transient GUS expression observed in targeted calli. Vacuum treatment during infection of calli with A. tumefaciens strains increased transformation efficiency. The highest stable transformation efficiency of transgenic plants was obtained when mature seed-derived calli infected with A. tumefaciens EHA101/pIG121Hm in the presence of 100 $\mu$M acetosyringone and vacuum treatment for 30 min. Southern blot analysis indicated integration of the transgene into the genome of tall fescue. The transformation system developed in this study would be useful for Agrobacterium-mediated genetic transformation of tall fescue plants with genes of agronomic importance.