• FLOWER RESEARCH JOURNAL
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• v.18 no.1
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• pp.1-8
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• 2010

Sensitivities of PLBs of four Phalaenopsis cultivars, P. 'Taisuco Windian', P. 'Nancy Amour', P. 'Pink Twilight' and P. 'Taipei Gold' to kanamycin, spectinomycin and hygromycin at different concentrations (0, 25, 50, 100, 200, and $400mg{\cdot}L^{-1}$) were examined. Hygromycin was favorable for selecting the transformants in the genetic transformation of Phalaenopsis as PLBs of four cultivars were all dead at even $25mg{\cdot}L^{-1}$ hygromycin. Responses of PLBs of P. 'Maki Watanabe' and P. 'Brother Lawrence' to DL-phosphinothricin (PPT) were determined at different concentrations (0, 0.1. 0.25, 0.5, 1.0, 2.0, 2.5, and $5.0mg{\cdot}L^{-1}$) and $0.5mg{\cdot}L^{-1}$ PPT was thought to be suitable for selecting the transformants of Phalaenopsis. The optimum conditions for Agrobacterium cocultivation with Phalaenopsis PLBs were examined using a two-step cocultivation method in Dtps. 'City Girl' and A. tumefaciens LBA4404. In the first infection period in a 1 : 10 suspension of Agrobacterium to a VW medium, 1 hr infection showed the highest PLB survival ratio. And then, PLBs were cocultivated with a bacterial strain and a 3-day cocultivation period was better for Phalaenopsis PLBs than a prolonged period. Agrobacterium tumefaciens strains LBA4404 (pTOK233) and EHA105 (pGA643) were used to compare their efficiency on the genetic transformation of Phalaenopsis PLBs. The PLBs infected with EHA105 survived more than those infected with LBA4404 after two days in a dark condition and two weeks in light condition on a selective medium. About 1,000 PLBs for each of P. 'Maki Watanabe' and P. 'Brother Lawrence', and each bacterial strain of AGL1 (pCAMBIA3301) and LBA4404 (pTOK233) were used for the regeneration of transgenic plants. The bacterial strain AGL1 had a higher genetic transformation efficiency than LBA4404, with no significant difference between cultivars. In this study, 11 hygromycin-resistant plantlets and 32 PPT-resistant plantlets were produced, but these putative transgenic plantlets need further examinations.

• Journal of Practical Agriculture & Fisheries Research
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• v.25 no.4
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• pp.138-147
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• 2024

Agrobacterium-mediated transformation (AMT) is a method that allows for the stable integration of DNA fragments into the plant genome. Transgenic plants generated through AMT typically exhibit a lower copy number of the transgene compared to those induced by particle bombardment. Furthermore, AMT offers a straightforward and efficient approach for generating transgenic plants. While the transformation efficiency of wheat is comparatively lower than that of other monocot plants such as Rice (Oryza sativa L.) and Maize (Zea mays L.), the cultivars 'Bobwhites' and 'Fielder' are commonly employed for wheat transformation. To date, there have been no reported instances of successful development of transgenic plants using Korean wheat varieties through AMT. This study aims to assess the transformation efficiency of 43 Korean wheat cultivars using the GUS assay, with the goal of identifying suitable Korean wheat cultivars for AMT. The pCAMBIA1301 vector, carrying the β-glucuronidase (GUS) gene, was incorporated into Agrobacterium strain EH105. Following the inoculation of Agrobacterium into immature embryos, GUS assays were conducted 'Saeol', 'Jopum', and 'Jonong' showed 100% (the number of embryos showing GUS spots/the number of embryos used for AMT) among 43 cultivars. In addition, cultivars with more than 70% were 'Saekeumgang', 'Jojung', 'Tapdong', 'Anbaek', 'Dabun', 'Sugang', 'Keumgang', 'Jeokjung', 'Seodun', 'Joeun', 'Dajung', and 'Baekjung'. It seems that the 15 cultivars above showed the possibility of using AMT. On the other hand, 'Yeonbaek', 'Goso', 'Baekgang', and 'Johan' showed less than 20% and GUS spots were not observed in 'Gru', 'Gobun', 'Milseong', and 'Shinmichal-1'. This study explores transient GUS expression in Korean wheat cultivars seven days after AMT. The observed initial high efficiency of transient transformation suggests the potential for subsequent stable transformation efficiency. Korean wheat cultivars demonstrating elevated transient transformation efficiency could serve as promising candidates for the development of stable transgenic wheat.

• Journal of Microbiology and Biotechnology
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• v.14 no.2
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• pp.390-394
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• 2004

The genetically modified glyphosate-tolerant soybean contains the following introduced DNA sequences: the EPSPS (5-enol-pyruvylshikimate-3-phosphate synthase) gene from Agrobacterium sp. strain CP4, the 35S promoter from the cauliflower mosaic virus, and the NOS terminator from Agrobacterium tumefaciens. In the present study, detection of these introduced DNAs was performed by amplification using the polymerase chain reaction (PCR). A multiplex PCR method was also applied to prevent false positive results. When primers for 35S promoter, nos3', CTP(chloroplast transit peptide), and CP4 EPSPS (EPSPS from Agrobacterium sp. CP4) were used, positive results were obtained in PCR reactions using DNA from genetically modified glyphosate-tolerant soybeans. There were no false positive results when using DNA from non-genetically modified soybeans. The CP4 EPSPS gene was detected when less than 125 pg glyphosate-tolerant soybean DNA was amplified. Lectin Lel and psb A were amplified from both non-genetically modified and genetically modified glyphosate-tolerant soybean DNA. Multiplex PCR was performed using different primer sets for actin Sacl, 35S promoter and CP4 EPSPS. The actin gene was detectable in both non-genetically modified and glyphosate-tolerant soybeans as a constant endogenous gene. Target DNAs for the 35S promoter, and CP4 EPSPS were detected in samples containing 0.01-0.1% glyphosate-tolerant soybean, although there were variations depending on primers by multiplex PCR. Soybean seeds from five plants of non-genetically modified soybean were co-cultivated for six months with those of genetically modified soybean, and they were analyzed by PCR. As a result, they were not positive for 35S promoter, nos3' or CP4 EPSPS. Therefore, these results suggest there was no natural crossing of genes between glyphosate-tolerant and non-genetically modified soybean during co-cultivation, which indicates that gene transfer between these plants is unlikely to occur in nature.

• Plant Biotechnology Reports
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• v.4 no.4
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• pp.253-260
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• 2010

Plant secondary metabolites have always been a focus of study due to their important roles in human medicine and nutrition. We transferred the isoflavone synthase (IFS) gene into soybean [Glycine max (L.) Merr.] using the Agrobacterium-mediated transformation method in an attempt to produce transformed soybean plants which produced increased levels of the secondary metabolite, isoflavone. Although the trial to produce transgenic plant failed due to unestablished hygromycin selection, transformed callus cell lines were obtained. The induction rate and degree of callus were similar among the three cultivars tested, but light illumination positively influenced the frequency of callus formation, resulting in a callus induction rate of 74% for Kwangan, 67% for Sojin, and 73% for Duyou. Following seven to eight subcultures on selection media, the isoflavone content of the transformed callus lines were analyzed by high-performance liquid chromatography. The total amount of isoflavone in the transformed callus cell lines was three- to sixfold higher than that in control callus or seeds. Given the many positive effects of isoflavone on human health, it may be possible to adapt our transformed callus lines for industrialization through an alternative cell culture system to produce high concentrations of isoflavones.

• Journal of The Korean Society of Grassland and Forage Science
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• v.25 no.4
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• pp.267-274
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• 2005

To develop transgenic orchardgrass (Dactylis glomerata L.) resistant to high temperature, a thermptolerance gene, DgP23, was introduced into orchardgrass using Agrobacterium - mediated transformation method. PCR and Southern blot analyses using genomic DNA showed specific DNA band on agarose gel and hybridization signal on X- ray film in transgenic orchardgrass harboring the recombinant DgP23 gene, but not in the wild type and empty vector control plants. RT-PCR and Southern blot analyses using total RNA also showed specific DNA band and hybridization signal. Transgenic orchardgrass did not showed ny morphological aberration both in the green house and field cultivation. Thermotolerance of transgenic plants was not detected in laboratory test. but may detected in field test.

• Journal of The Korean Society of Grassland and Forage Science
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• v.29 no.4
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• pp.299-306
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• 2009

Oxidative stress is the main limiting factor in crop productivity. To solve global environmental problems using the plant biotechnology, we have developed on the oxidative stress-tolerant transgenic tall fescue plants via Agrobacterium-mediated genetic transformation method. In order to develop transgenic tall fescue (Festuca arundinacea Schreb.) plants with enhanced tolerance to multiple environmental stresses, nucleotide diphosphate kinase gene under the control of CaMV35S promoter were introduced into genome of tall fescue plants. Proteomic analysis revealed that transgenic tall fescue not only accumulated NDP kinase 2 protein in their cells, but also induced several other antioxindative enzyme-related proteins. When leaf discs of transgenic plants were subjected to cold stress, they showed approximately 30% less damage than wild-type plants. In addition, transgenic tall fescue plants showed normal growth when transgenic plants were subjected to $4^{\circ}C$ for 3 days treatments. These results suggest that transgene is important in ROS scavenging by induction of antioxidative proteins, and could improve abiotic stress tolerance in transgenic tall fescue plants.

• 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.

• Molecules and Cells
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• v.41 no.5
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• pp.413-422
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• 2018

Soybean transgenic plants with ectopically expressed AtABF3 were produced by Agrobacterium-mediated transformation and investigated the effects of AtABF3 expression on drought and salt tolerance. Stable Agrobacterium-mediated soybean transformation was carried based on the half-seed method (Paz et al. 2006). The integration of the transgene was confirmed from the genomic DNA of transformed soybean plants using PCR and the copy number of transgene was determined by Southern blotting using leaf samples from $T_2$ seedlings. In addition to genomic integration, the expression of the transgenes was analyzed by RT-PCR and most of the transgenic lines expressed the transgenes introduced. The chosen two transgenic lines (line #2 and #9) for further experiment showed the substantial drought stress tolerance by surviving even at the end of the 20-day of drought treatment. And the positive relationship between the levels of AtABF3 gene expression and drought-tolerance was confirmed by qRT-PCR and drought tolerance test. The stronger drought tolerance of transgenic lines seemed to be resulted from physiological changes. Transgenic lines #2 and #9 showed ion leakage at a significantly lower level (P < 0.01) than ${\underline{n}}on-{\underline{t}}ransgenic$ (NT) control. In addition, the chlorophyll contents of the leaves of transgenic lines were significantly higher (P < 0.01). The results indicated that their enhanced drought tolerance was due to the prevention of cell membrane damage and maintenance of chlorophyll content. Water loss by transpiration also slowly proceeded in transgenic plants. In microscopic observation, higher stomata closure was confirmed in transgenic lines. Especially, line #9 had 56% of completely closed stomata whereas only 16% were completely open. In subsequent salt tolerance test, the apparently enhanced salt tolerance of transgenic lines was measured in ion leakage rate and chlorophyll contents. Finally, the agronomic characteristics of ectopically expressed AtABF3 transgenic plants ($T_2$) compared to NT plants under regular watering (every 4 days) or low rate of watering condition (every 10 days) was investigated. When watered regularly, the plant height of drought-tolerant line (#9) was shorter than NT plants. However, under the drought condition, total seed weight of line #9 was significantly higher than in NT plants (P < 0.01). Moreover, the pods of NT plants showed severe withering, and most of the pods failed to set normal seeds. All the evidences in the study clearly suggested that overexpression of the AtABF3 gene conferred drought and salt tolerance in major crop soybean, especially under the growth condition of low watering.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.5
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• pp.657-662
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• 2008

To develop transgenic orchardgrass (Dactylis glomerata L.) resistant to high temperature, the recombinant DgHSP17.2 gene was introduced into orchardgrass plants using the Agrobacterium-mediated transformation method and expressed constitutively under the control of the CaMV 35S promoter. The results of genomic DNA PCR and Southern analysis showed a DNA band and hybridization signal on agarose gel and X-ray film in transgenic orchardgrass plants harboring the recombinant DgHSP17.2 gene, but a DNA band and hybridization signal were not observed in the wild type and empty vector control plants. The same result was also obtained in RT-PCR and Southern blot analysis, and these transgenic orchardgrass plants did not show any morphological aberration both in the culture bottle and soil mixture. When leaf discs cut from transgenic orchardgrass plants with recombinant DgHsp17.2 gene were exposed to lethal temperature (heat treatment at $60^{\circ}C$ for 50 min), 60-80% of the leaf discs showed only damage symptoms, but non-transgenic leaf discs showed a lethal condition. These results indicate that the DgHsp17.2 gene may act as a protector from heat stress in plants.

• Horticultural Science & Technology
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• v.34 no.2
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• pp.314-323
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• 2016

A transient ihpRNA-induced gene silencing system based on Agrobacterium-mediated injection infiltration has been established to evaluate candidate genes involved in proanthocyanidin (PAs) biosynthesis in persimmon (Diospyros kaki Thunb.). We chose DkPDS (phytoene desaturase) as a gene-silencing target to evaluate the newly developed transient gene silencing system. Our qRT-PCR analysis indicated that two ihpRNA constructs (pHG-PDS5' and pHG-PDS3') targeted DkPDS, which also led to significantly reduce expression of DkPDS in 'Mopanshi' persimmon leaves. To further confirm the reliability of the system, we successfully utilized it for DkLAR (leucoanthocyanidin reductase) gene silencing. The expression levels of DkLAR in 'Mopanshi' and 'Eshi 1' leaves were ca. 6-fold and ca. 5-fold lower than those in leaves harboring empty vector (pHG-GFP), respectively. DMACA (4-dimethylaminocinnamaldehyde) staining and the Folin-Ciocalteau assay showed that the accumulation of PAs was markedly inhibited in 'Mopanshi', 'Eshi 1' and 'Youhou' leaves. These results indicate that DkLAR plays an important role in the accumulation of PAs in persimmon. The transient ihpRNA-induced gene silencing method developed in this study is a highly efficient and useful tool for functional analysis of persimmon genes involved in PA biosynthesis.