• Korean Journal of Weed Science
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• v.30 no.3
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• pp.225-232
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• 2010

The effect of Protox expression site on herbicidal resistance was investigated in wild-type and transgenic rice plants imposed by peroxidizing herbicide oxyfluorfen. The transgenic rice systems involved the plastidal expression of Arabidopsis protoporphyrinogen oxidase (Protox; AP line) and the dual expression of Myxococcus xanthus Protox in chloroplasts and mitochondria (TTS line). The oxyfluorfen-treated TTS4 line showed the lower levels of cellular leakage and malonyldialdehyde and the sustained capacity of 5-aminolevulinic acid synthesis, compared to the oxyfluorfen-treated AP and wild-type lines. During oxyfluorfen action, the TTS4 line had greater herbicide resistance than the AP1 line, indicating that the dual expression of M. xanthus Protox in chloroplasts and mitochondria prevented the accumulation of photodynamic protoporphyrin IX more effectively than the expression of Arabidopsis Protox only in chloroplasts. These results suggest that the ectopic expression of Protox in mitochondria greatly contributes to the herbicidal resistance in rice plants.

• Journal of Plant Biotechnology
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• v.37 no.4
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• pp.483-493
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• 2010

Knock-out of a gene by insertional mutagenesis is a direct way to address its function through the mutant phenotype. Among ca. 15,000 gene-trapped Ds insertion lines of rice, we identified one line from selected sensitive lines in highly salt stress. We conducted gene tagging by TAIL-PCR, and DNA gel blot analysis from salt sensitive mutant. A gene encoding an oligopeptide transporter (OPT family) homologue was disrupted by the insertion of a Ds transposon into the OsOPT10 gene that was located shot arm of chromosome 8. The OsOPT10 gene (NP_001062118.) has 6 exons and encodes a protein (752 aa) containing the OPT family domain. RT-PCR analysis showed that the expression of OsOPT10 gene was rapidly and strongly induced by stresses such as high-salinity (250 mM), osmotic, drought, $100\;{\mu}M$ ABA. The subcellular localization assay indicated that OsOPT10 was localized specifically in the plasma membrane. Overexpression of OsOPT10 in Arabidopsis thaliana and rice conferred tolerance of transgenic plants to salt stress. Further we found expression levels of some stress related genes were inhibited in OsOPT10 transgenic plants. These results suggested that OsOPT10 might play crucial but differential roles in plant responses to various abiotic stresses.

• Research in Plant Disease
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• v.18 no.1
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• pp.24-28
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• 2012

The rice diseases occurring on the transgenic rice plant carrying protox gene (CPPO06) was assessed and compared with other varieties of rice such as Dongjin, Chucheong, Ilpeum, and Onnuri in the fields located in Cheongwon of Chungbuk, Suwon of Gyeunggi, and Gwangju of Chonnam. In the field of Cheongwon, the diseases such as blast, leaf spot, sheath blight and Fusarium blight were observed. False smut were observed only in the field of Suwon, where the ratio of diseased plants was 0.28% in CPPO06 and 0.37% in Onnuri, respectively. In the field of Gwangju, leaf spot caused by Cochlioborus miyabeanus was the most severely occurring disease among rice diseases mentioned above. Fusarium blight occurred in all the 3 fields, which were more severe in CPPO06 plants treated with or without oxadiazon as the herbicide. Except for Fusarium blight, there was no significant difference in the rice diseases as blast, leaf spot, sheath blight and false smut between CPPO06 and other rice varieties.

• The Plant Pathology Journal
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• v.28 no.4
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• pp.364-372
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• 2012

Xanthomonas oryzae pv. oryzae causing bacterial leaf blight disease in rice produces and secretes Hpa1 protein that belongs to harpin protein family. Previously it was reported that Hpa1 induced defense responses when it was produced in tobacco. In this study, we expressed hpa1 gene in rice and Arabidopsis to examine the effects of Hpa1 expression on disease resistance to both fungal and bacterial pathogens. Expression of hpa1 gene in rice enhanced disease resistance to both X. oryzae pv. oryzae and Magnaporthe grisea. Interestingly, individual transgenic rice plants could be divided into four groups, depending on responses to both pathogens. hpa1 expression in Arabidopsis also enhanced disease resistance to both Botrytis cineria and Xanthomonas campestris pv. campestris. To examine genes that are up-regulated in the transgenic rice plants after inoculation with X. oryzae pv. oryzae, known defense-related genes were assessed, and also microarray analysis with the Rice 5 K DNA chip was performed. Interestingly, expression of OsACS1 gene, which was found as the gene that showed the highest induction, was induced earlier and stronger than that in the wild type plant. These results indicate that hpa1 expression in the diverse plant species, including monocot and dicot, can enhance disease resistance to both fungal and bacterial plant pathogens.

• 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 Microbiology and Biotechnology
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• v.19 no.8
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• pp.781-786
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• 2009

Small interfering synthetic double-stranded RNA (siRNA) was applied to suppress the expression of the human cytotoxic-T-Iymphocyte antigen 4-immunoglobulin (hCTLA4Ig) gene transformed in transgenic rice cell cultures. The sequence of the 21-nucleotide siRNA was deliberately designed and synthesized with overhangs to inactivate the expression of hCTLA4Ig. The chemically synthesized siRNA duplex was combined with polyethyleneimine (PEl) at a mass ratio of 1:10 (0.33 ${\mu}g$ siRNA:3.3 ${\mu}g$ PEl) to produce complexes. The siRNA complexes (siRNA+PEI) were labeled with Cy3 in order to subsequently confirm the delivery by fluorescent microscopy. In addition, the cells were treated with sonoporation at 40 kHz and 419W for 90 s to improve the delivery. The siRNA complexes alone inhibited the expression of hCTLA4Ig to 45% compared with control. The siRNA complexes delivered with sonoporation downregulated the production of hCTLA4Ig to 73%. Therefore, we concluded that the delivery of siRNA complexes into plant cells could be enhanced successfully by sonoporation.

• Rapid Communication in Photoscience
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• v.3 no.2
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• pp.28-31
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• 2014

Loss of chlorophyll is the visible symptom of leaf senescence and staygreen refers to the delayed leaf senescence in plants. The staygreen gene (SGR) in rice (Oryza sativa L.) has been identified as its mutation maintains greenness during leaf senescence, and encodes a chloroplast protein required for the initiation of chlorophyll breakdown in plants. In this study, we isolated a rice SGR-homologous gene in creeping bentgrass (Agrostis stolonifera L.), and transgenic creeping bentgrass plants were obtained by introducing pCAMBIA3301 vector harboring antisense SGR gene under control of the senescence-specific SAG12 promoter. Transgenic plants were selected by herbicide resistance assays and genomic integration of the transgenes was confirmed by PCR analysis. Subsequent analyses demonstrated the staygreen phenotype of the transgenic creeping bentgrass plants with decreased chlorophyll loss during leaf senescence. These results suggest that the antisense SGR expression in creeping bentgrass delays leaf senescence, which provides a way to develop genetically engineered turfgrass varieties with the commercially useful staygreen trait.

• Korean Journal of Plant Tissue Culture
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• v.22 no.3
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• pp.175-182
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• 1995

Rice is one of the most successful monocot in regenerating fertile and genetically stable transgenic plants. However there is no report of a rice line developed in Korea that can be used for regeneration of fertile and genetically stable transformants. In this paper we first demonstrate that a Korean variety Nakdongbyeo, is suitable to obtain transgenic rice plants. Protoplasts from embryogenic suspension cultures were co-transformed with HPT (hygromycin phosphotransferase) and GUS ($\beta$-glucuronidase) genes in separate plasmids in the presence of PEG (polyethylene glycol). In 5 independent experiment, the average frequency of calli showing hygromycin resistance were 1.73%. Plantlets were regenerated from the Hy $g^{R}$ calli. The average efficiency of plantlet regeneration was apprbximately 27%. Based on the GUS activities of hygromycin resistant calli, ca.35% of the resistant calli carried active GUS genes. The R0 transgenic plantlets were grown to maturity and Rl seeds were obtained. By examining the in siぉ activity of GUS in Rl seeds and seedlings, we confirmed that the GUS transgene driven by a CaMV 35S (cauliflower mosaic virus) promoter showed proper expression patterns. We also confirmed Mendelian segregation of the HPT transgene in the Rl generation.n.

• Korean Journal of Breeding Science
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• v.41 no.3
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• pp.252-260
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• 2009

Rice is the most important cereal crop not only in supplying the basic staple food for more than half of the world's population but also as a model plant for functional genomic studies of monocotyledons. Although rice transformation method using A. tumefaciens has already been widely used to generate transgenic plants, the transformation rate is still low in most Korean elite cultivars. We made several modifications of the standard protocol especially in the co-cultivation step to improve the efficiency of the rice transformation. The co-culture medium was modified by the addition of three antioxidant compounds (10.5 mg/L L-cysteine, 1 mM sodium thiosulfate, 1 mM dithiothreitol) and of Agrobacterium growth-inhibiting agent (5 mg/L silver nitrate). Co-cultivation temperature ($23.5^{\circ}C$ for 1 day, $26.5^{\circ}C$ for 6 days) and duration (7 days) were also changed. The plasmid of pMJC-GB-GUS carrying the GUS reporter gene and the bar gene as the selectable marker was used to evaluate the efficiency of the transformation. After co-cultivation, a high level of GUS gene expression was observed in calli treated with the modified method. It is likely that those newly added compounds helped to minimize the damage due to oxidative bursts during plant cell-Agrobacterium interaction and to prevent necrosis of rice cells. And the transformation rate under the modified method was also remarkably increased approximately 8-fold in Heungnambyeo and 2-fold in Ilmibyeo as compared to the corresponding standard method. Furthermore, we could produce the transgenic plants stably from Ilpumbyeo which is a high-quality rice but its transformation rate is extremely low. Transformation and the copy number of transgenes were confirmed by PCR, bar strip and Southern blot analysis. The improved method would attribute reducing the effort and the time required to produce a large number of transgenic rice plants.

• Journal of Life Science
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• v.24 no.6
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• pp.618-625
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• 2014

Rice flour is used in many food products. However, dough made from rice lacks extensibility and elasticity, making it less suitable than wheat for many food products such as bread and noodles. The high-molecular weight glutenin subunits (HMW-GS) of wheat play a crucial role in determining the processing properties of the wheat grain. This paper describes the development of marker-free transgenic rice plants expressing a wheat Glu-Dy10 gene encoding the HMG-GS from the Korean wheat cultivar 'Jokyeong' using Agrobacterium-mediated co-transformation. Two expression cassettes, consisting of separate DNA fragments containing Glu-1Dy10 and hygromycin phosphotransferase II (HPTII) resistance genes, were introduced separately into Agrobacterium tumefaciens EHA105 for co-infection. Each EHA105 strain harboring Glu-1Dy10 or HPTII was infected into rice calli at a 3: 1 ratio of Glu-1Bx7 and HPTII. Among 290 hygromycin-resistant $T_0$ plants, we obtained 29 transgenic lines with both the Glu-1Dy10 and HPTII genes inserted into the rice genome. We reconfirmed the integration of the Glu-1Dy10 gene into the rice genome by Southern blot analysis. Transcripts and proteins of the Glu-1Dy10 in transgenic rice seeds were examined by semi-quantitative RT-PCR and Western blot analysis. The marker-free plants containing only the Glu-1Dy10 gene were successfully screened in the $T_1$ generation.