• Korean Journal of Plant Tissue Culture
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• v.21 no.5
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• pp.309-314
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• 1994

To develop simple and efficient transformation methods of monocotyledonous plane, electroporation-mediated delivery of DNA into intact embryogenic cell clumps was investigated in zoysiagrass and rice. Calli of zoysiagrass, induced from 3-week-old immature embryos, were suspension-cultured in MS basic medium supplemented with 1.0 mg/t 2.4-D and used for elechuporation. Calli, derived from immature inflorescences of 20 mm lenth of rice, were also suspension-cultured on N6 basic medium supplemented with 1.0 mg/L 2.4-D. Suspension-cultured embryogenic cell clumps were electroporated in liqid MS medium added with a Plasmid DNA (30 $\mu\textrm{m}$/ml), pGA1074, encoding ${\beta}$-glucuronidiase (GUS). DNA delivery into the cells through cell walls and cell membrane was confirmed by the transient expression of the GUS gene. Cell clumps of zoysiagrass and rice, electroporated with 400 volt at 800 pF capacitance, expressed GUS gene activity at a mean frequency of 25 units (one unit = one clony of blue cells) per 200 ${\mu}\ell$ of packed cell volume. Untreated cells and healed non-embryogenic cells did not exhibit GUS activity These results indicate that electroporation-mediated transformation can use intact embryogenic cells (thus avoiding the use protoplasts) in zoysiagrass and rice.

• Journal of Plant Biotechnology
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• v.35 no.3
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• pp.169-176
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• 2008

Salicylic acid(SA) is a phytohormone that is related to plant defense mechanism. The SA accumulation is triggered by abiotic and biotic stresses. SA acts as a signal molecular compound mediating systemic acquired resistance and hypersensitive response in plant. Although the role of SA has been studied extensively, an understanding of the SA regulatory mechanism is still lacking in plants. In order to comprehend SA regulatory mechanism, we have been transformed with a SID2 promoter:GUS::LUC fusion construct into siz1-2 mutant and wild plant(Col-0). SIZ1 encodes SUMO E3 ligase and negatively regulates SA accumulation in plants. SID2(SALICYLIC ACID INDUCTION DEFICIENT2) is a crucial enzyme of SA biosynthesis. The Arabidopsis SID2 gene encodes isochorismate synthase(ICS) that controls SA level by conversion of chorismate to isochorismate. We compared the regulation of SID2 in wild-type and siz1-2 transgenic plants that express SID2 promoter:GUS::LUC constructs respectively. The expressions of $\beta$-GLUCURONIDASE and LUCIFERASE were higher in siz 1-2 transgenic plant without any stress treatment. SID2 promoter:GUS::LUC/siz1-2 transgenic plant will be used as a starting material for isolation of siz1-2 suppressor mutants and genes involved in SA-mediated stress signaling pathway.

• Horticultural Science & Technology
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• v.33 no.6
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• pp.947-954
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• 2015

To select suitable spray chrysanthemum cultivars for Agrobacterium-mediated transformation, thirty-nine (39) spray cultivars bred in the National Institutes of Korea and a standard cultivar Jinba from Japan were collected and tested for regeneration rate and Agrobacterium infection assays. MS medium with $0.5mg{\cdot}L^{-1}$ IAA and $1.0mg{\cdot}L^{-1}$ BAP was used for shoot regeneration from leaf disks and internodes. The shoot regeneration rate in leaf disks was the highest in cultivar BRM, followed by cultivars VS, WW and YTM. The cultivar JB (Jinba) used as a transformation material in previous reports ranked similarly to cultivars PK and SPP. In shoot regeneration from internodes, the shoot regeneration rate was the highest for cultivar PA, followed by cultivar WW. The infection rate of leaves and internodes of 40 chrysanthemum cultivars with agrobacterium was investigated. Cultivars WPP, YNW, VS, PP, WW, FA, PA and YMN showed the highest infection levels in leaves, whereas cultivars WPP, PA, PK and YNW had the highest infection levels in internodes. Considering all of these results, cultivars VS and WW were the most appropriate for gene transformation of chrysanthemum using leaves, while cultivar PA was for internodes.

• Horticultural Science & Technology
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• v.18 no.5
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• pp.594-598
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• 2000

This study was carried out to investigate the tissue-specific expression of ${\beta}$-glucuronidase (gus) gene driven by endosperm-specific promoter (Z4 promoter) in the transgenic tobacco and to find out inheritance pattern of transgene to the next generation. Tobacco (Nicotiana tabaccum cv. Havana SR1) was transformed with Agrobacterium tumerfaciens LBA4404 harboring BV3 construct containing gus gene driven by Z4 promoter and a kanamycin resistant gene. Seven hundred bp PCR products, indicating the presence of npt II gene, were found in the all eight transformants by PCR analysis using nptII primers. To study the expression pattern of the two different kind of promoters, leaf disks of the Z4pro-gus-transformed plants and 35Spro-gus-transformed plants were analyzed histochemically for gus activity. As a result, leaf disks of Z4pro-gus-transformed plants showed very weak and partial positive gus activity. In contrast, leaf disks of 35Spro-gus-transformed plants showed relatively strong positive gus activity. To investigate the expressed position of Z4 promoter, seeds from Z4pro-gus-transformed plants and 35Spro-gus-transformed plants were analyzed histochemically for gus activity. Z4pro-gus-transformed seeds showed positive gus activity restricted to the endosperm. However, the blue-colored product in 35Spro-gus-transformed seeds was observed in all the area including endosperm. Kanamycin resistance assay showed that transgenes were stably inherited to next generation in all lines.

• 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.4
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• pp.193-198
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• 2006

Effects of acetosyringone and on Agrobacterium-mediated transformation of orchardgrass were investigated. Embryogenic calli induced from 3 varieties, Frontier, Potomac and Roughrider, 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 varieties and acetosyringone(AS) concentrations on transformation and the expression of the GUS gene were investigated. Inclusion of $200{\mu}M$ AS in inoculation and co-cultivation media lead to a significant increase in stable transformation efficiency. Hygromycin resistant calli were developed into complete plants via somatic embryogenesis. GUS histochemical assay and PCR analysis of transgenic plants demonstrated that transgenes were integrated into the genome of orchardgrass.

• Korean Journal of Plant Tissue Culture
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• v.25 no.1
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• pp.13-19
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• 1998

We have reported previously that intact embryogenic cells can be used instead of protoplasts for electroporation-mediated transformation of zoysiagrass and rice. In this study, conditions of the tissue electroporation were examined to optimize the procedures. Embryogenic cell suspensions were established in liquid MS medium containing 2 mg/L of 2,4-D with embryogenic calluses induced from mature embryos of Z. japonica. The suspension-cultured cell clumps were electroporated with 35S-gusA expression vector DNA, and degrees of DNA introduction into the cells were determined by histological expression rates of the gusA marker gene. DNA transfer into the cell clumps occurred in wide range of voltage (100-400 V) and capacitance (10-1980 $\mu\textrm{F}$), but more in the ranges of 200-300 V and 330-800 $\mu\textrm{F}$ DNA concentrations higher than 6 $\mu\textrm{g}$/mL were adequate for GUS expression of the electroporated cells. DNA transfers were confirmed in all three embryogenic cell lines but only in one out of eleven non-embryogenic lines. Positive GUS expressions occurred with DNAs added even 20-40 h after pulse treatments. As a promoter of gusA, Act1 and Ubi1 were effective 7 and 5 times than 35S respectively in number of GUS expression units on electroporated cell clumps. Embryogenic cell clumps survived and regenerated into plantlets after pulse treatments of wide range of conditions.

• Journal of Plant Biotechnology
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• v.33 no.1
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• pp.49-56
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• 2006

Gerber (Gerbera hybrida) is a valuable ornamental species grown as a potted plant and cut flowers. However, genetic variability within the gerbera genus is very limited. So it is absolutely needed to introduce and widen genetic resources into gerbera lines by genetic transformation. For the purpose, 18 Korean gerbera lines were screened to establish Agrobacterium-mediated genetic transformation procedure. In an experiment to select Korean gerbera lines which are amenable to Agrobacterium-inoculation, 12 lines turned out to be positive in Agrobacterium-inoculation. More callus were produced from BA 2ppm, Zeatin 2ppm, IAA 0.2ppm in pre-culture and regeneration medium (2X media) but there was no difference in the frequency of GUS expression rate. In another experiment to find out optimal condition for highly efficient Agrobacterium-inoculation, petiole and leaf explants have been treated with four different pre-culture periods, two different co-culture periods and two different Agrobacterium strains. As a result, high GUS expression has been showed from petiole and leaf explants treated no pre-culture period with LBA4404 Agrobacterium tumerfaciens, 5 day co-culture period and dipping treatment.

• Journal of Plant Biotechnology
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• v.2 no.2
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• pp.61-71
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• 2000

By screening a cDNA library of auxin-treated mung bean (Vigna radiata L.) hypocotyls, we have isolated two full-length cDNA clones, pVR-ACS6 and pVR-ACS7, for 1-aminocyclopropane-1-carboxylate (ACC) synthase, the rate-limiting enzyme in the ethylene biosynthetic pathway. While PVR-ACS6 corresponds to the previously identified PCR fragment pMBA1, pVR-ACS7 is a new cDNA clone. A comparison of deduced amino acid sequences among auxin-induced ACC synthases reveal that these enzymes share a high degree of homology (65-75%) to VR-ACS6 and VR-ACS7 polypeptides, but only about 50% to VR-ACS1 polypeptide. ACS6 and ACS7 are specifically induced by auxin, while ACS1 is induced by cycloheximide, and to lesser extent by excision and auxin treatment. Results from nuclear run-on transcription assay and RNA gel blot studies revealed that all three genes were transcriptionally active displaying unique patterns of induction by IAA and various hormones in etiolated hypocotyls. Particularly, 24-epibrassinolide (BR), an active brassinosteroid, specifically enhanced the expression of VR-ACS7 by distinct temporal induction mechanism compared to that of IAA. In addition, BR synergistically increased the IAA-induced VR-ACS6 and VR-ACS7 transcript levels, while it effectively abolished both the IAA- and kinetin-induced accumulation of VR-ACS1 mRNA. In light-grown plants, VR-ACS1 was induced by IAA in roots, whereas W-ACS6 in epicotyls. IAA- and BR-treatments were not able to increase the VR-ACS7 transcript in the light-grown tissues. These results indicate that the expression of ACC synthase multigene family is regulated by complex hormonal and developmental networks in a gene- and tissue-specific manner in mung bean plants. The VR-ACS7 gene was isolated, and chimeric fusion between the 2.4 kb 5'-upstream region and the $\beta$-glucuronidase (GUS) reporter gene was constructed and introduced into Nicotiana tobacum. Analysis of transgenic tobacco plants revealed the VR-ACS7 promoter-driven GUS activity at a highly localized region of the hypocotyl-root junction of control seedlings, while a marked induction of GUS activity was detected only in the hypocotyl region of the IAA-treated transgenic seedlings where rapid cell elongation occurs. Although there was a modest synergistic effect of BR on the IAA-induced GUS activity, BR alone failed to increase the GUS activity, suggesting that induction of VR-ACS7 occurs via separate signaling pathways in response to IAA and BR.

• Journal of Plant Biotechnology
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• v.32 no.4
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• pp.257-262
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• 2005

Agrobacterium tumefaciens-mediated cotyledonary-node explants transformation was used to produce transgenic melon. Cotyledonary-node explants of melon (Cucumis melo L. cv. Super VIP) were co-cultivated with Agrobacterium strains (LBA4404, GV3101, EHA101) containing the binary vector (pPTN289) carrying with CaMV 35S promoter-gus gene as reporter gene and NOS promoter-bar gene conferring resistance to glufosinate (herbicide Basta) as selective agent, and the binary vector (pPTN290) carrying with Ubiquitin promoter-GUS gene and NOS promoter-nptll gene conferring resistance to paromomycin as selective agent, respectively. The maximum transformation efficiency (0.12%) was only obtained from the cotyledonary-node explants co-cultivated with EHA101 strain (pPTN289) on selection medium with 5 mg/L glufosinate and not produced a transgenic melon from the cotyledon or cotyledonary-node co-cultivated with other strains. Finally, five plants transformed showed the resistance in glufosinate antibiotic and the GUS positive response in leaf ($T_0$), flower ($T_0$), seeds ($T_1$) and plantlet ($T_1$). Southern blot analysis revealed that the gus gene integrated into each genome of transgenic melon.