• 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.

• 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.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.37 no.1
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• pp.102-109
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• 2010

Fructan has been found to accumulate in various tissues during periods when light levels increased carbon fixation where low temperatures reduced growth rates while photosynthesis continued. In this study, we have cloned 1-sucrose:sucrose fructosyl transferase(1-sst) and 1-fructan: fructan fructosyl transferase (1-fft, a key enzyme for the synthesis of fuctan) from Jerusalem Artichoke (Helianthus tuberosus L.). The recombinant vector with 1-sst and 1-fft has been constructed under the control of 35S promoter of KJGV-B2 vector and transgenic plants obtained by Agrobacterium tumefaciens LBA4404. PCR analysis carried out on the putative transgenic plants for amplification of the coding region of specific gene (1-sst, 1-fft), and HPT genes. Transgenic lines carrying of 1-sst and 1-fft were confirmed for integration into the rice genome using Southern blot hybridization and RT-PCR. The transgenic plants in $T_2$ generation were selected and expression pattern analysis revealed that 1-sst and 1-fft were stable. This analysis confirmed the presence of low-molecular-weight fructan in the seedling of the transgenic rices. Therefore, cold tolerance and carbohydrate metabolism will be possible to develop resistant plants using the transgenic rice.

• Journal of The Korean Society of Grassland and Forage Science
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• v.27 no.2
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• pp.109-116
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• 2007

Environmental stress is the major limiting factor in plant productivity. As an effort to solve the global food and environmental problems using the plant biotechnology, we have developed transgenic tall fescue (Festuca arundinacea Schreb.) plants via Agrobacterium-mediated gene transfer method. To develop transgenic tall fescue plants with enhanced tolerance to the environmental stresses, both CuZn superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX) genes were incorporated in a pIG121 binary vector and the both of the genes were controlled separately by an oxidative stress-inducible sweet potato peroxidase 2 (SWPA2) premoter expressed in chloroplasts. Leaf discs of transgenic plants showed 10-30% less damage compared to the wild-type when they exposed to a wide range of environmental stresses including methyl viologen (MV), $H_2O_2$ and heavy metals. In addition, when $200{\mu}M$ MV was sprayed onto the whole plants, transgenic plants showed a significant reduction of visible damage compared to wild-type plants that were almost damaged. These results suggest that over expression of CuZnSOD and APX genes in transgenic plants might be a useful strategy to protect the crops against a wide range of environmental stresses.

• The Korean Journal of Ecology
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• v.28 no.2
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• pp.93-98
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• 2005

The effects of the presence of a submerged plant, Zizania latifolia, on physico-chemical characteristics, including Eh, pH, and concentrations of nitrogen and phosphorus in the sediments were studied under pot culture condition. It was shown that Eh value at reduced layer of the sediments was higher in the planted pots than in the non-planted. It was also revealed that $NH_4^+-N$ concentration of the sediments in the planted pots was lower than that of the non-planted, which might be due to the uptake by the plants. In contrast, $NO_3^--N$ concentration in the sediment increased in the presence of the plants compared to the non-planted, which might be attributed to oxygen released from the roots to the reduced layer. The concentration of organic phosphorus in the sediments was much higher than that of NAIP at the beginning of the planting experiment. However, at the end of the experiment, it was reversed; NAIP concentration was much higher than that of organic phosphorus, possibly indicating the transformation of organic phosphorus to NAIP during the experimental period. Both concentrations of $NH_4^+-N$ and $PO_4^{3-}-P$ in the overlying and percolated water were lower in the planted pots than in the non-planted. The concentration of $NO_3^--N$ in the percolated water, however, was higher in the planted pots than in the non-planted. The data was discussed with regard to the potential effects of a submerged plant on dynamics of phosphorus and nitrogen in the rhizosphere of the sediment.

• Applied Biological Chemistry
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• v.38 no.3
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• pp.224-231
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• 1995

To increase the expression of a foreign protein in transgenic plant, the benefits of 5'-untranslated leader sequences of tobacco mosaic virus (TMV) RNA or soybean glycinin gene, Gy2, fused to a protein coding sequence were exploited. pGA643-derived plasmid contains 355 promoter of cauliflower mosaic virus, protein coding sequence of maize 10 kDa zein (10kZ) and Gy2 terminator. The leader from Gy2 or TMV RNA was inserted between the promoter and the coding sequence in each construct. The recombinant DNAs were introduced into tobacco plants by Agrobacterium mediated leaf disc transformation method. Although the transgene without the leader had more transcripts than the others, mRNAs containing the leader were translated more efficiently. It might be due to difference in the length of 5'-untranslated sequence and context surrounding the AUG codon, but could be sequence specific rather. These results suggest that the leader sequences of Gy2 and TMV play important roles as an enhancer in translational control of foreign gene in transgenic tobacco plant.

• Korean Journal of Plant Tissue Culture
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• v.25 no.2
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• pp.109-113
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• 1998

In this study we tried to transform an antimicrobial peptide gene (Shiva) under the promoter of tomato phenylalanine ammonia-lyase (tPAL5) into tobacco and potato plants. Antimicrobial peptide gene was isolated originally from giant silk moth (Hyalophora cecropia) and modified ie nucleotide sequence to increase antimicrobial activity. Transgenic tobacco plants were regenerated and their seeds were tested on the media containing kanamycin (500 mg/L). The results of PCR amplification and genomic Southern blot hybridization confirmed the integration of construct (tPAL5 promoter-Shiva-NOS-GUS-NOS) into chromosome. We observed that one of the transgenic tobacco plants showed chromosome rearrangement when integrated. In case of potato transformation, the efficiency of regeneration was maximized at the medium containing Zeatin 2mg/L, NAA 0.01mg/L, GA$_3$ 0.1mg/L. We also observed the high expression of GUS (${\beta}$-glucuronidase) enzyme which was located next to the terminator sequence of nopaline synthase gene (NOS) in the vascular tissue of stem, leaves of transgenic potatoes. This result suggested that a short sequence of Shiva gene (120 bp) and NOS terminator sequence might be served as a leader sequence of transcript when translated.

• Korean Journal of Plant Tissue Culture
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• v.25 no.3
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• pp.177-181
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• 1998

This study was carried out to investigate activity difference in the superoxide dismutase (SOD) and peroxidase (POD) of tomato callus transformed with Agrobacterium containing the GUS gene. Than those of other two tomato cultivars, the hypocotyl explant of JA101 was shown to have higher POD and SOD specific activity of 23 unit/mg protein and 2,156 unit/mg protein, respectively. Relatively high frequency of callus formation was obtained from the hypocotyl explant on MS medium containing 1 mg/L 2,4-D for 30 days and its POD(47 unit/mg protein) and SOD (95,786 unit/mg protein) specific activities were higher than other 2,4-D concentration. The hypocotyl explant and callus cocultivated with Agrobacterium for 72 hours were transferred to MS medium supplemented with 1 mg/L 2,4-D, 30 mg/L kanamycin, 30 g/L sucrose and 4 g/L Gelrite. The hypocotyl explants transferred to the medium formed callus with 45.5% effeciency after 8 weeks. The transformation efficiency confirmed by GUS assay was 21.6%. POD specific activity of the transformed callus (54 unit/mg protein) were somewhat lower than the non-transformed callus (64 unit/mgg protein) and SOD specific activity of the transformed callus (30,300 unit/mg protein) were also lower than the non-transformed callus (37,077 unit/mg protein). However there was no significant difference in POD and SOD isozyme patterns between the transformed and the non-transformed calluses. From these results, it revealed that there was no difference of antioxidant enzyme activities between the transformed callus and the non-transformed callus in tomato.

• Korean Journal of Plant Tissue Culture
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• v.25 no.4
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• pp.283-288
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• 1998

The bialaphos is a potent inhibitor of glutamine synthease in higher plants and is used as a non-selective herbicide. We have used the bialaphos resistant gene(Bar) encoding for an acetyltransferase isolated from Streptomyces hygroscopicus SF1293. Callus derived from mature seeds of rice(Oryza sativa L. cv. Dong Jin) were co-cultivated with Agrobacterium tumefaciens EHA101 carring a plasmid pGPTV-HB containing genes for hygromycin resistance (HygR) and Bar. Transgenic plants showing in vitro resistance to 50 mg/L hygromycin and 10 mg/L bialaphos were obtained by using a two-step selection/regeneration procedure. Transformation efficiency of rice was about 30% which was as high as reported in other dicotyledons. Progenies ($\textrm{T}_{1}$ generation) derived from primary transformant of 17 lines were segregated with a 3 resistant : 1 sensitive ratio in medium containing hygromycin and bialaphos. Stable integration of Bar gene into chromosomal DNA was proven by Southern blot analysis of genomic DNA isolated from $\textrm{T}_{2}$ progenies. Transgenic plants ($\textrm{T}_{3}$) grown in the field were resistant to bialaphos (Basta) at a dosage lethal to wild type plants.