• Proceedings of the Korean Society of Grassland Science Conference
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• 2005.06a
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• pp.158-159
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• 2005

• Proceedings of the Korean Society of Grassland Science Conference
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• 2005.06a
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• pp.174-175
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• 2005

• Journal of The Korean Society of Grassland and Forage Science
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• v.29 no.3
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• pp.165-170
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• 2009

A system for the production of transgenic plants has been developed for perennial ryegrass (Lolium perenne L.) via Agrobacterium-mediated transformation. Included in this study were two factors which may affect the gene transfer efficiency: concentrations of acetosyringone (AS, 0 to 300 ${\mu}M$), and co-culture period (1 to 7 days). Both factors were very important to achieve high efficiency gene transformation in the perennial ryegrass. The highest transformation efficiency was obtained when embryogenic calli were inoculated with Agrobacterium in the presence of 100 ${\mu}M$ AS with the culture medium for 5 days. Phosphinothricin resistant calli were developed with into complete plants. GUS histochemical assay, polymerase chain reaction (PCR) and Northern blot analysis of transgenic plants demonstrated that transgenes were integrated into the genome of perennial ryegrass. Using this protocol, it was possible to obtain transformants efficiently for further study.

• Journal of The Korean Society of Grassland and Forage Science
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• v.17 no.4
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• pp.379-386
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• 1997

Recombinant plasmid, pBKH4, containing NPT II and P35S-BcHSP17.6 was constructed by ligation of Bum H I -digested pBKSl-l and BcHSP 17.6 (thermotolerance gene) 6om pBLH4. The tobacco leaf disc was cocultivated with transformed Agmbacterium tumefaciens bearing pBKH4 for 24 hours and transformed shoots were selected on MS-n/B medium containing $100\;{\mu\textrm{g}}/ml$ of kanamycin. Heat-killing temperature of Nicotima tabacum was $50^{\circ}$ for >15min, and transformed tobacco plants with BcHSP17.6 cDNA exhibited thermotolerance at the heat-killing temperature. The transgenic plants were analyzed by Southern blot hybridization with the probe of ${\alpha}^{_32}P$ labelled BcHSP17.6 cDNA. Transcription and expression level of BcHSP17.6 cDNA were also continued by Northern blot analysis and Ouchterlony double immunodiffusion assay. In this study, we suggest that the BcHSP17.6 cDNA introduced to tobacco plant is related to thenuoto-lerance and 17.6-kD LMW HSP acts as a protector from heat damage in plants.

• Proceedings of the Korean Society of Crop Science Conference
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• 2022.10a
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• pp.234-234
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• 2022

Gene-editing is currently one of the most popular technologies in recent years. Development of the new crop using the gene editing have advantage of improved accuracy and efficiency compared with conventional breeding. Soybean (Glycine max L.) is one of the most important crops worldwide used as food and forage. We tried to establish a system for breeding improvement of soybean through gene-editing technology. For the gene-editing system of soybean, i) selection of efficiency gRNA of targeted gene, ii) efficient genetic transformation of the selected gRNA, iii) selection of trans-clean mutant is essential. First of all, we investigated the selection conditions of gRNA with high editing efficiency of targeted gene using isolated protoplast of soybean. Furthermore, we performed the Agrobacterium-mediated genetic transformation of various soybean cultivars. We identified the tissue culture ability in 23 soybean cultivars for genetic transformation of soybean. The six cultivars with high tissue culture ability were selected and confirmed the transgenic plants in four cultivars. Finally, we established a speed-breeding system as a powerful tool for the fast selection of trans-clean mutants from transgenic plants. Our laboratory will provide the valuable system for improvement of soybean by the gene-editing technology.

• Journal of The Korean Society of Grassland and Forage Science
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• v.26 no.4
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• pp.187-192
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• 2006

In order to investigate the effects of genetic variations of orchardgrass in tissue culture response, calli were induced from mature seeds of eight varieties, 'Hapsung 2', '93E', 'Amba', 'Ambassdor', 'Frode', 'Frontier', 'Potomac' and 'Roughrider', and plant regeneration frequency was compared. Significant differences were observed among the varieties in both callus induction and plant regeneration. Callus induction rate of viable seeds varied from 24.3% to 71.7%. Plant regeneration frequency ranged from 76.6% to 29.7%. 'Roughrider' varieties showed higher regenerability with the frequency of 76.6%. These results can be used not only to provide additional improvements in the plant regeneration frequency from transgenic callus, but also useful for molecular breeding of orchardgrass through genetic transformation.

• Journal of The Korean Society of Grassland and Forage Science
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• v.17 no.3
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• pp.285-292
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• 1997

This study was conducted to obtain the transgenic Brnssica napus plants with tobacco Apase gene using the binary vector system of Agrobacteriurn fumefociens. The results obtained were summarized as follows: A repressible acid phosphatase gene of Saccharon~yces cerevisiae, pho105 was used for screening of tobacco Apase cDNA. In order to identify Apase gene in tobacco genome, Southern blot analysis was pcrformed and the Apase gcnc may be present as a single copy, or at most two or three copies, in tobacco genome. To isolate the tobacco Apase gene, tobacco cDNA library was constructed using purifed mRNA from -Pi treated tobacco root and the plaque forming unit of the library was 2.8 x $10^5$ pfu/m${\ell}$, therefore the library might cover all expressed mRNAs. Using pho5 as a probe. tobacco Apase cDNA was cloned, and restriction mapping and Southern blot analysis of cDNA insert were revealed that the 3.6 kb cDNA contained tobacco acid phosphatase cDNA. Plasmid pGA695 -tcAPl was constructed by subcloning tobacco Apase cDNA into the Hind site of pGA695 with 35s promoter which can be expressed constitutively in plants. The Brassica napus cotyledonary petioles were cocultivated with the ,4 grobacteriunz and transferred to the selection medium. The transformed and regenerated plants were transplanted to soil medium. Southern blot analysis was done on the transformed plants, and it was confirmed that a foregin gene was stably integrated into the genonies of B. nnpus plants.

• Journal of The Korean Society of Grassland and Forage Science
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• v.31 no.3
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• pp.229-234
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• 2011

In order to develop an efficient, reliable and reproducible tissue culture system for reed (Phragmites communis Trinius), an efficient plant regeneration system via callus induction was established using mature seeds as explants. MS medium containing 1 mg/L 2,4-D and 0.5 mg/L BA was optimal for callus induction from mature seeds. The highest frequency (88.7%) of callus formation was obtained in 1.0 mg/L 2,4-D. The highest plant regeneration frequency (59.6%) was found when the embryogenic calli were subcultured on MS medium supplemented with 100 mg/L myo-inositol, whereas, adding of plant growth regulators was not so promising in this case. Our result would be useful for development of transgenic reed plants.

• Journal of The Korean Society of Grassland and Forage Science
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• v.28 no.3
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• pp.165-170
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• 2008

Timothy (Phleum pratense L.) is an important grass species as forage. In order to optimize tissue culture conditions of timothy, the effects of plant growth regulators on callus induction and plant regeneration was investigated with mature seeds of colt cultivar. The optimal concentration of 2,4-D for the induction of primary callus from mature seeds was 3 mg/L. The highest embryogenic callus frequenc (25%) was observed when the mature seed were cultured on MS medium supplemented with 3 mg/L 2,4-D and 0.1 mg/L BA. The highest plant regeneration frequency was observed when type B callus was transferred to N6 medium supplemented with 1 mg/L 2,4-D and 3 mg/L BA. Regenerated plants were grown normally when shoots were transplanted to the soil. A short tissue culture period and regeneration system would be beneficial for molecular breeding of timothy by the production of transgenic plant.

• Journal of The Korean Society of Grassland and Forage Science
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• v.33 no.2
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• pp.100-104
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• 2013

Zoysiagrass (Zoysia japonica Steud.) is an important forage and turfgrass that spreads by stolons and rhizomes. Zoysiagrass stolon can be used directly for Agrobacterium-mediated genetic transformation by exploiting the potential of direct shoot formation. However, surface sterilization of field-grown stolons is difficult and remains to be explored. We developed an effective surface sterilization and culture method using the stolon explant for infection with Agrobacterium tumefaciens. Among various treatments, sequential disinfection in 30% bleach for 15 min followed by 0.1% mercuric chloride for 25 min resulted in the highest number of clean stolons. The efficacy of mercuric chloride was increased under vacuum conditions by incubating at 800 mbar for 5 min. The inclusion of 2.5 mg/l amphotericin B further prevents fungal growth in in vitro cultures. This protocol would speed up the development of transgenic plants by utilizing field-grown stolon nodes.