• Journal of Plant Biotechnology
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• v.40 no.1
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• pp.37-42
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• 2013

Rice is one of the most important cereal crops as a model plant for functional genomics of monocotyledons and usually transformed using Agrobacterium tumefaciens. However, the transformation's process using previous method is still time consuming and uneconomical, low efficiency. In this study, we established a new method by modifying the general Agrobacterium protocol especially in the infection and co-cultivation, Agrobacterium elimination, infected calli's selection steps using liquid media. We directly inoculated Agrobacterium containing a ZjLsL gene under the control of constitutive promoter into the 1- to 3-week-old rice calli derived from mature seeds. After 3 days of co-cultivation, the infected calli were transferred onto liquid media of Agrobacterium elimination and calli's selection for 3 days. The calli were transferred to calli's growth solid media for 14 days and then the calli transferred to shoot induction and root induction media. Putative transformants were initially selected on the medium containing phosphinothricin, and the PAT protein verified by PAT strip test. This method in this study would lead to reduction of substantial labor and time to generate transgenic plants.

• Journal of Plant Biotechnology
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• v.38 no.2
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• pp.105-116
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• 2011

Transgenic zoysiagrass (Zoysia japonica Steud.) expressing the bar gene inserted in the plant genome has been generated previously through Agrobacterium tumefaciens-mediated transformation. The GM zoysiagrass (event: JG21) permits efficient management of weed control of widely cultivated zoysiagrass fields, reducing the frequency and cost of using various herbicides for weed control. Now we have carried out the environmental risk assessment of JG21 prior to applying to the governmental regulatory agency for the commercial release of the GM turf grass outside of test plots. The morphological phenotypes, molecular analysis, weediness and gene flow from each test plot of JG21 and wild-type zoysiagrasses have been evaluated by selectively analyzing environmental effects. There were no marked differences in morphological phenotypes between JG21 and wild-type grasses. The JG21 retained its stable integration in the host plant in T1 generation, exhibiting a 3:1 segregation ratio according to the Mendelian genetics. We confirmed the copy number (1) of JG21 by using Southern blot analysis, as the transgenic plants were tolerant to ammonium glufosinate throughout the culture period. From cross-fertilization and gene flow studies, we found a 9% cross-pollination rate at the center of JG21 field and 0% at distances over 3 m from the field. The JG21 and wild-type zoysiagrass plants are not considered "weed" because zoysiagrasses generally are not dominant and do not spread into weedy areas easily. We assessed the horizontal gene transfer (HGT) of the transgene DNA to soil microorganisms from JG21 and wild-type plants. The bar gene was not detected from the total genomic DNA extracted from each rhizosphere soil of GM and non-GM Zoysia grass fields. Through the monitoring of JG21 transgene's unintentional release into the environment, we found no evidence for either pollen mediated gene flow of zoysiagrass or seed dispersal from the test field within a 3 km radius of the natural habitat.

• Korean Journal of Breeding Science
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• v.40 no.3
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• pp.278-285
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• 2008

An efficient transformation method for soybean [Glycine max (L.) Merr.] using meristematic tissues of germinating seeds has been established. The embryonic axes were excised from germinating seeds of Korean soybean cultivar, Iksannamulkong and 0.5-2 cm long segment containing meristematic tissues were prepared by cutting hypocotyl region. The explants were inoculated with Agrobacterium tumefaciens strain LBA4404 harboring a binary vector with the bar gene as a selectable marker gene and a ${\beta}-glucuronidase$ (GUSINT) reporter gene, and then co-cultured for 7 days on co-cultivation medium (CCM). The meristematic tissues were cultured on shoot induction medium (SIMP6) supplemented with 0.4 mg/l $N_6-benzylaminopurine$ (BAP) and 0.1 mg/l indolebutyric acid (IBA) in the presence of 6 mg/l L-phosphinotricin (PPT) for 2 weeks and the surviving explants were transferred to shoot elongation medium (SEMP6). Transformation was confirmed by Southern blot analysis and the transformation efficiencies ranged from 1.48 to 2.07%. The new modified transformation method was successfully implemented for obtaining several transgenic lines with SMV-CP gene. It is expected that this method could efficiently be used for the transformation of recalcitrant soybean cultivars.

• Korean Journal of Medicinal Crop Science
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• v.15 no.4
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• pp.285-290
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• 2007

This study was conducted to introduce phosphinothricin acetyl transferase (PAT) gene, resistant to basta which was non-selective herbicide, into balloon flower (Platycodon grandiflorum A. De. candolle). Seeds were germinated on MS medium, and 10-day-old immature cotyledon explants and 30-day-old leaf explants were cocultured with Agrobacterium tumefaciens strain MP 90 (pBinSyn) on 1/10 MS medium for 48 hours in the dark at $25^{\circ}C$. The cultures were transferred for selection of kanamycin-resistant shoots to the MS medium supplemented with 0.2 $mg/{\ell}$ NAA, 1.0 $mg/{\ell}$ BA, 3% sucrose, 100 $mg/{\ell}$ kanamycin, 500 $mg/{\ell}$ carbenicillin. Shoots were obtained from 10-day-old immature cotyledon explants after 4 weeks of culture. The shoots were subcultured twice every 4 weeks on the same medium for growth of transgenic shoots. Successful transformation was confirmed by histochemical GUS assay, PCR analysis, RT-PCR analysis, 10 $mg/{\ell}$ phosphinothricin treatment and 0.3% basta spray. The basta-resistant transgenic plants flowered normally.

• Journal of Plant Biotechnology
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• v.42 no.3
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• pp.180-185
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• 2015

Leaf discs from 'Yeobong' and 'Maehyang' strawberry plants were used as explants for transformation. The Agrobacterium tumefaciens strain EHA105 harboring the monellin gene under the control of the CaMV 35S promoter was used in co-cultivation experiments. The frequencies of callus formation and plant regeneration from leaf explants after co-cultivation in 'Yeobong' were higher than those of 'Maehyang'. These transgenic plants showed normal growth patterns and flowering. PCR and Southern hybridization confirmed that 1 to 2 copies of the monellin gene were integrated into genome of the transgenic strawberry plants. Northern blot analysis confirm that the transcripts were expressed in transgenic strawberry plants. Although long-term subcultured transgenic strawberry plants showed a phenomenon to escape the transgene, the transformation system established in this study provides new opportunities for genetic improvement of strawberry plants.

• Applied Biological Chemistry
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• v.37 no.5
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• pp.343-348
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• 1994

Ti plasmid of A. tumefaciens was labeled with $^3H-thymidine$, purified and encapsulated into phosphatidylserine (PS) and PS-cholesterol (Chol; 1 : 1 molar ratio) liposomes by lyophilization-rehydration method. PS was supplemented with 1 mole percent octadecyl rhodamine B for fluorometric measurement of PS. Liposomes entrapping $^3H-Ti plasmid$ were fused with Nicotiana sanderae protoplasts by treating with 5 mM $CaCl_2$ and 10% PEG. The fusion was evidenced by fluorescence microscopic technique. The amounts of Ti plasmid and PS associated with protoplasts were assayed by the radioactivity of $^3H-Ti plasmid$ and by the fluorescence of rhodamine B. About 7.9% of the PS liposome and 7.2% of PS-Chol liposome were fused with protoplasts. During the fusion process, about 30% of the liposomal contents of PS-Chol liposome was leaked, in contrast to about 60% leakage of its contents in PS liposome. Accounting the number of liposomes fused with protoplasts together with the encapsulation efficiency and the leakage of liposomal contents, it was calculated that ca. 1,700 Ti plasmid was transfered into one protoplast by the present method. This result may indicates that the present method transfers enough Ti plasmid into plant protoplast to elicit genetic transformation of plants.

• Korean Journal of Plant Tissue Culture
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• v.25 no.3
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• pp.147-152
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• 1998

AL1-gene, necessary for the replication of the genome of a gemini virus TGMV, was inserted in the opposite direction to the promoter CaMV35S resulting in the construction of a plant transformation binary vector pAR35-2. The vector pAR35-2 contains the chimeric gene cassette involving the duplicated promoter CaMV35S, opposite direction of AL1-gene fusioned with hygromycin resistant gene, and the gene cassette of the neomycin phosphotransferase II gene. The plasmid was transferred to tobacco and tomato plants by leaf disk infection via Agrobacterium. The transgenic plants were selected and grown on the MS-agar medium containing kanamycin and hygromycin. The shoots induced from the calli were regenerated to the whole transgenic plants. The antisense AL1-gene was detected in the genomic DNA isolated from the leaves by using the PCR mediated Southern blot analysis. The expression of the antisense AL1-gene was also observed using the RT-PCR mediated Southern blot analysis. The observation of chloroplasts in guard cell pair indicated that the transgenic tomato plants were diploid.

• 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.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.

• Korean Journal of Plant Tissue Culture
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• v.24 no.1
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• pp.63-69
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• 1997

Erythropoietin (EPO) is a glycoprotein that mediates the growth and differentiation of erythroid progenitors. In order to produce recombinant human erythropoietin in tobacco plant, the EPO genomic DNA (5.4 kb) was cloned into plant expression vectors, pBI$\Delta$GUS121, pBD$\Delta$GUS121 and pPEV-1, and introduced in Nicotiana tabacum (var. Xanthi) via Agrobacterium tumefaciens-mediated transformation. After selection on MS media containing kanamycin (Km), 10 Km-resistant plants were obtained per each construct. The correct integration of EPO genomic DNA in the genome of transgenic plant was confirmed by polymerase chain reaction (PCR). Northern blot showed that transcripts of 1.8 kb length were produced in leaves of the plants, but there was no difference of mRNA amount according to promoter number and 5'-untranslated sequence (UTS). The proteins obtained from leaves of transgenic plants were immunologically detected by Western blot using rabbit anti-human EPO polyclonal antibody. The expressed protein appeared as smaller band of apparent mass of 30 kDa as compared to the EPO protein from human urine (37 kDa), suggesting that the modification (glycosylation) system in tobacco plant might be different from that of mammalian cells.