• Horticultural Science & Technology
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• v.29 no.4
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• pp.345-351
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• 2011

This study was carried out to develop a model system using selection method for disease resistant plant breeding programs using a herbicide bialaphos-resistant patII gene as a gene-based marker. Spraying bialaphos could eliminate the susceptible plants from the segregating populations such as ${F_2}^{\prime}s$ and thereafter. Tomato cv. Momotaro-yoke was transformed with patII gene 60 independent transformants were acquired. Total 42 transformants were analyzed in transgene copy numbers by Southern blotting and the segregation ratios for the bialaphos resistance. Statistical analysis revealed that the transgene copy numbers and the segregation ratios were not always coincided, especially having the tendency of underestimating the real numbers of the transgenes in the multicopy lines. A two-stepwise screening method was applied to select $T_1$ tomato plants which linked the transgenic patII to a disease resistance gene (I2 and Ve). Based on the resistant to susceptible ratios, T-20 plant was finally selected due to the estimated linkage 12-13 cM between the patII gene to the I2 gene on chromosome 11. This newly developed system could be applied to any economical crop in breeding programs.

• 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.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.49 no.3
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• pp.237-242
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• 2004

A system for the production of transgenic plants has been developed for tall fescue (Festuca arundinacea Schreb.) via Agrobacterium-mediated transformation of mature seed-derived embryogenic callus. Seed-derived calli were infected and co-cultured with Agrobacterium EHA101 carrying standard binary vector pIG121Hm encoding the hygromycin phosphotransferase (HPT), neomycin phosphotransferase II (NPTII) and intron-containing $\beta$-glucuronidase (intron-GUS) genes in the T-DNA region. The effects of several factors on transformation and the expression of the GUS gene were investigated. Inclusion of $200\mu\textrm{M}$ acetosyringone (AS) in inoculation and co-culture media lead to a increase in stable transformation efficiency. Transformation efficiency was increased when embryogenic calli were co-cultured for 5 days on the co-culture medium. The highest transformation efficiency was obtained when embryogenic calli were inoculated with Agyobacterium in the presence of 0.1% Tween20 and $200\mu\textrm{M}$ AS. Hygromycin resistant calli were developed into complete plants via somatic embryogenesis. GUS histochemical assay and Southern blot analysis of transgenic plants demonstrated that transgenes were successfully integrated into the genome of tall fescue.

• Journal of The Korean Society of Grassland and Forage Science
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• v.26 no.1
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• pp.1-8
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• 2006

To develop a new variety of orchardgrass with improved digestibility, caffeic acid O-methyltransferase (Dgcomt), which is a methylation enzyme involved in the early stages of lignin biosynthesis, was isolated and characterized. Dgcomt was expressed not only in leaves but also in stems and roots. The expression levels of transcripts were high in stems and roots which are the most lignified tissues, and only moderate levels of transcripts were expressed in leaves. To develop transgenic orchardgrass plants by down-regulating the Dgcomt gene, an RNAi suppression vector with partial Dgcomt DNA fragment was constructed and transferred into the genome of orchardgrass via Agrobacterium-mediated gene transfer method. PCR and Southern blot analyses with genomic DNAs from putative transgenic plants revealed that the T-DNA region containing RNAi construct was successfully integrated into the genome of orchardgrass. Northern blot analysis revealed that the majority of the down-regulated transgenic lines showed significant reduction in Dgcomt gene expression. These RNAi transgenic orchardgrass will be useful for molecular breeding of new variety with improved digestibility by down-regulating lignin biosynthetic enzyme.

• Journal of The Korean Society of Grassland and Forage Science
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• v.26 no.2
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• pp.77-82
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• 2006

An efficient transformation system for the production of transgenic plants has been developed for tall fescue (Festuca arundinacea Schreb.) via Agrobacterium-mediated transformation of seed-derived callus. From the point of morphogenetic capacity, three types of callus were selected. High frequency of plant regeneration was obtained by selection of type II callus, and the plant regeneration frequency was 52.6% when embryogenic callus were cultured on the regeneration medium. Supplementation of the media with 10 mg/L $AgNO_3$ and 40 mg/L cysteine enhanced frequencies of plant regeneration up to 65.3%. The highest transformation efficiency was also obtained when type II callus were inoculated with Agrobacterium. Southern blot analysis of PCR products of transgenic plants demonstrated that transgenes were successfully integrated into the genome of tall fescue. Efficient regeneration system and transformation established in this study will be useful for molecular breeding of tall fescue through genetic transformation.

• Korean Journal of Microbiology
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• v.41 no.2
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• pp.140-145
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• 2005

Streptomyces natalensis ATCC27448 produces natamycin, a commercially important macrolide antifungal antibiotic. For molecular genetic study of S. natalensis, we have developed a system for introducing DNA into S. natalensis via conjugal transfer from Escherichia coli. An effective transformation procedure for S. natalensis was established based on transconjugation from E, coli ET12567/pUZ8002 using a ${\Phi}C31$-derived integration vector, pSET152, containing oriT and attP fragments. The high frequency was obtained on MS medium containing 10 mM $MgCl_2$ using $6.25\times10^8$ of E.coli donor cells without heat treatment of spores. In addition, southern blot analysis of exconjugants and the sequence of plasmids containing DNA flanking the insertion sites from the chromosome revealed that S. natalensis contains a single ${\Phi}C31$ attB site and at least a secondary or pseudo attB site. Similar to the case of various Streptomyces species, a single ${\Phi}C31$ attB site of S. natalensis is present within an ORF encoding a pirin-homolog, but a pseudo-attB site is present within a distinct site (GenBank accession no. $YP\_117731$) and also its sequence deviates from the consensus sequences of attB sequence.

• Journal of Life Science
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• v.19 no.3
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• pp.378-383
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• 2009

Agrobacterium tumefaciens-mediated transformation procedure for the phalaenopsis orchid, established by using Protocorm-like bodies (PLBs), was aimed at the introduction of target genes into individuals with divergent genetic backgrounds. PLBs obtained from the axillary bud of a peduncle were maintained on a hyponex medium supplemented with 1 g/l of activated charcoal, 30 g/l of sucrose and 0.1 mg/l thiamine. The multiplication rate of PLBs was about 90% in case of subculture PLBs to be cut transversely into 1/3 part from top position. The PLBs were inoculated with Agrobacterium strain EHA105 harboring both $\beta$-glucuronidase (GUS) and hygromycin-resistant genes for 20 minutes after dipping treatment. Transformation efficiency was the highest with a Agrobacterium culture medium and dipping treatment of O.D. 0.8. Newly induced PLBs were put on selection medium containing 1 mg/l hygromycin for 2 months. Hygromycin-resistant phalaenopsis plants that regenerated after the selection culture of PLBs showed histochemical blue staining due to GUS. Transgene integration of the hygromycin-resistant plants was confirmed by PCR and Southern blot using GUS specific primers and probe.

• Journal of Plant Biotechnology
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• v.34 no.1
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• pp.37-45
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• 2007

The genetic transformation of watermelon by Agrobacterium has been known very difficult and a few successful cases have been reported by obtaining the direct shoot formation. However, since this direct shoot formation is not guaranteed the stable transformation, the stable transformation with reproducibility is required by a different approach such as a callus induced manner. The best conditions for inducing the callus from cotyledon and root explants of watermelon were 2 mg/L zeatin + 0.1 mg/L IAA and 2 mg/L BA + 0.1 mg/L 2,4-D, respectively. The GFP expression in the callus was identified and monitored through fluorescent microscopy after transformation with pmGFP5-ER vector. Paromomycin rather than kanamycin was used for selecting the nptll gene expression because it was more effective to select the watermelon explants. Four different callus types were observed and the solid green callus showed stronger GFP expression. The highest frequency of GFP expression in the callus developed from cotyledon was 9.0% (WM8 inbred line), while the highest frequency from root was 8.3% (WM6 inbred line). The WMV-CP was transformed using the method of GFP transformation and the genetic transformation of WMV-CP was confirmed by PCR and Southern blot analysis. Here we present a system for callus induction of watermelon explant and the callus induced method would facilitate the establishment of stable watermelon transformation.

• Journal of Sericultural and Entomological Science
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• v.38 no.1
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• pp.42-47
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• 1996

To investigate the host range determining factors of nuclear polyhedrois virus (NPV), Autographa california NPV and Bombyx mori NPV were coinfected into the two different cell lines, BmN-4 and Sf-9. The recombinant baculoviruses, RecS-A6 and RecB-727 which have an expanded host range, were isolated from Sf-9 and BmN-4 cell lines, respectively. The molecular biological characteristics of the recombinant baculoviruses were investigated. The pathogenicity of RecB-727 was similar to that of wild type BmNPV, while the pathogenicity of RecS-A6 was relatively lower than that of wild type BmNPV. The restriction enzyme digestion patterns of parental viruses and recombinant viruses showed that the recombinant virus has an expanded host range by genetic recombination. Southern blot analysis revealed that the p10 gene of RecB-727 was derived from AcNPV genomic DNA, while RecS-A6 has p10 gene of BmNPV in a viral genome. To investigate the host range expansion mechanism of recombinant baculovirus, HindIII-SacI 0.6 kb DNA fragments of RecS-A6 and RecB-727 were cloned and sequenced. The results showed that of wild type BmNPV helicase gene, suggesting that the expanded host range of recombinant baculoviruses was due to the insertion of BmNPV helicase gene into AcNPV viral genome.

• Journal of Plant Biotechnology
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• v.34 no.4
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• pp.277-283
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• 2007

Embryogenic calluses derived from shoot apical meristem explants of sweetpotato were subjected to particle bombardment to generate transgenic plants for antisense expression of cDNAs encoding two different AGPase small subunit (ibAGP1 and ibAGP2). Plants were generated via somatic embryogenesis. PCR and Southern analysis demonstrated that the incorporation of ibAGP1 and ibAGP2 into the genome in an antisense orientation. Immunoblot analysis confirmed reduced levels of AGPase small subunit in transgenic plant leaves. Plants with both ibAGP1 and ibAGP2 produced a lower level of the protein than plants with ibAGP1 alone. iodine test demonstrated that transgenic plant leaves and storage root accumulated reduced amounts of starch. Iodine staining of leaf tissues indicated that transgenic plants accumulated less amount of starch than control. In accordance with western blot analysis, plants with both ibAGP1 and ibAGP2 accumulated a lower amount of starch than plants with ibAGP1 alone. Both transgenic plants exhibited a severely retarded growth, resulting in bare survival. It is suggested that disrupted expression of the gene encoding AGPase small subunit is lethal to the growth of sweetpotato contrast to other species including potato.