• 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.

• 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.

• Journal of Plant Biotechnology
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• v.37 no.3
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• pp.319-326
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• 2010

An interest in the production of seed-oil based fuel and raw materials, which comes from renewable plant sources, has been intrigued by the phenomenon of global warming and shortage of fossil fuels. Rapeseed (Brassica napus) is the most important oilseed crop, which produces seeds with 40% oil. It is desirable to develop genetically modified rapeseed producing oils, which can be easily converted to biodiesel. As an initial step for development of genetically modified rapeseed for the production of biofuels or bio-based materials, Korean rapeseed cultivars, Naehan, Youngsan, Tammi and Halla, were analyzed. Four Korean rapeseed cultivars produce 32 to 40% oil of seed dry weight, which is rich in oleic acid (more than 60 mole%). The cotyledonary petioles of rapeseed cultivar, Halla, were transformed using Agrobacterium tumefaciens strain GV3101, carrying the uidA gene encoding $\beta$-glucuronidase (GUS) as a reporter gene and the phosphinothricin acetyltransferase (PAT) gene as a selectable marker. The stable integration of PAT gene in the genome of transgenic rapeseeds was confirmed by PCR analysis. Expression of uidA gene in various rapeseed organs was determined by fluorometric assay and histochemical staining. Transformation efficiency of a Korean rapeseed Halla cultivar was 10.4%. Genetic inheritance of transgenes was confirmed in $T_2$ generation.

• Journal of Plant Biotechnology
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• v.5 no.3
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• pp.163-168
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• 2003

A routine system based on particle bombardment of embryogenic callus for recovery of fertile transgenic rice (Oryza sativa L.) plants was developed. Embryogenic callus was established within 2-3 months from calli derived from mature seeds of Korean rice cultivar, Nagdongbyeo. The callus was bombarded with the plasmid pRQ6 containing the $\beta$-glucuronidase gene (gusA) and hygromycin phosphotransferase gene (hph, conferring resistance to hygromycin B), both driven by CaMV 35S promoter. Placement of cells on an osmoticum-containing medium (0.2 M sorbitol and 0.2 M mannitol) 4 hrs prior to and 16 hrs after bombardment resulted in a statistically significant increase with 3.2-fold in transient expression frequency gusA. In five independent experiments, the average frequency of transformation showing GUS activities was $8.86\%$. A large number of morphologically normal, fertile transgenic rice plants were obtained. Integration of foreign gene into the genome of $R_0$ transgenic plants was confirmed by Southern blot analysis. GUS and HPT were detected in $R_1$ progeny and Mendelian segregation of these genes was observed in $R_1$ progeny.

• Journal of Plant Biotechnology
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• v.4 no.4
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• pp.143-150
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• 2002

A strong oxidative stress-inducible peroxidase promoter (referred to as SWPA2 promoter) was cloned from tell cultures of sweetpotato (Ipomoea batatas) and characterized in transgenic tobacco cultured cells in terms of biotechnological applications. Employing a transient expression assay in tobacco protoplasts, with five different 5'-deletion mutants of the SWPA2 promoter fused to the $\beta$-glucuronidase (GUS) reporter gene, the 1314 bp deletion mutant showed approximately 30 times higher GUS expression than the CaMV 35S promoter. The expression of GUS activity in suspension cultures of transgenic cells derived from transgenic tobacco leaves containing the -1314 bp SWPA2 promoter-GUS fusion was strongly expressed following 15 days of subculture compared to other deletion mutants, suggesting that the 1314 bp SWPA2 promoter will be biotechnologically useful for the development of transgenic cell lines engineered to produce key pharmaceutical proteins. In this respect, we developed transgenic cell lines such as tobacco (Nicotiana tabacum L. BY-2), ginseng (Panax ginseng) and Siberian ginseng (Acanthopanax senticosus) using a SWPA2 promoter to produce a human lactoferrin (hLf) and characterized the hLf production in cultured cells. The hLf production monitored by ELISA analysis in transgenic BY-2 cells was directly increased proportional to cell growth and reached a maximal level (up to 4.3% of total soluble protein) at the stationary phase in suspension cultures. The SWPA2 promoter should result in higher productivity and increased applications of plant cultured cells for the production of high-value recombinant proteins.

• 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.

• Plant Biotechnology Reports
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• v.4 no.2
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• pp.129-138
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• 2010

An efficient transformation system for high-throughput functional genomic studies of kiwifruit has been developed to overcome the problem of necrosis in Actinidia arguta explants. The system uses Agrobacterium tumefaciens strain EHA105 harbouring the binary vector pART27-10 to inoculate leaf strips. The vector contains neomycin phosphotransferase (nptII) and ${\beta}$-glucuronidase (GUS) (uidA) genes. A range of light intensities and different strengths of Murashige and Skoog (MS) basal salt media was used to overcome the problem of browning and/or necrosis of explants and calli. Callus browning was significantly reduced, resulting in regenerated adventitious shoots when the MS basal salt concentration in the culture medium was reduced to half-strength at low light intensity ($3.4\;{\mu}mol\;m^{-2}\;s^{-1}$) conditions. Inoculated leaf strips produced putative transformed shoots of Actinidia arguta on half-MS basal salt medium supplemented with 3.0 $mg\;l^{-1}$ zeatin, 0.5 $mg\;l^{-1}$ 6-benzyladenine, 0.05 $mg\;l^{-1}$ naphthalene acetic acid, 150 $mg\;l^{-1}$ kanamycin and 300 $mg\;l^{-1}$ $Timentin^{(R)}$. All regenerated plantlets were deemed putativ transgenic by histochemical GUS assay and polymerase chain-reaction analysis.

• Plant Biotechnology Reports
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• v.2 no.3
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• pp.219-226
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• 2008

Transgenic plants with the herbicide-resistance gene (bar gene) were obtained via organogenesis from isolated mesophyll protoplasts of Nierembergia repens after applying electroporation. Transient ${\beta}-glucuronidase$ (GUS) activity of electroporated protoplasts assayed 2 days after applying an electric pulse showed that optimum condition (transient GUS activity 319 pmol 4 MU/mg per min and plating efficiency 2.43%) for electroporation was 0.5 kV/cm in field strength and $100{\mu}F$ in capacitance. The protoplasts electroporated with the bar gene at this condition initiated formation of microcolonies on medium after 2 weeks. After 4 weeks of culture, equal volume of fresh 1/2-strength Murashige and Skoog (MS) medium containing 0.2 mg/l bialaphos was added for selection of transformed colonies. After 6 weeks of culture, growing colonies were transferred onto regeneration medium containing 1.0 mg/l bialaphos, on which they formed adventitious shoots 1-2 months after electroporation. The adventitious shoots rooted easily after transfer onto MS medium with bialaphos lacking plant-growth regulators. Transformation of these regenerants with the bar gene was confirmed by Southern analysis. Some of the transformants showed strong resistance to the application of bialaphos solution at 10.0 mg/l.

• Korean Journal of Medicinal Crop Science
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• v.12 no.5
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• pp.424-427
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• 2004

Microsomal ${\omega}-3$ fatty acid desaturase (FAD3) is an essential enzyme in the production of the n-3 polyunsaturated fatty acid ${\alpha}-linolenic$ acid during the seed developing stage. To understand the regulatory mechanism of the gene encoding the ${\omega}-3$ fatty acid desaturase, a genomic fragment corresponding to the previously isolated perilla seed PfFAD3 cDNA was amplified from perilla (Perilla frutescens Britt) by GenomeWalker PCR. Sequence analysis of the fragment provided with identification of a 1485-bp 5'-upstream region and a 241-bp intron in the open reading frame. To determine the tissue-specificity of the PfFAD3 gene expression, the 5'-upstream region was fused to the ${\beta}-glucuronidase$ (GUS) gene and incorporated into Arabidopsis thaliana. Histochemical assay of the transgenic plants showed that GUS expression was restricted to seed and pollen, showing that PfFAD3 gene was exclusively expressed in those tissues.

• Journal of Plant Biotechnology
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• v.6 no.2
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• pp.69-75
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• 2004

Optimal protocol for efficient genetic transformation has been defined to aid future strategies of genetic engineering in pigeon pea with agronomically important genes. Transgenic pigeonpea plants were successfully produced through Agrobacterium tumefaciens-mediated genetic transformation method using cotyledonary node explants by employing defined culture media. The explants were co-cultivated with A. tumefaciens strain C-58 harboring the binary plasmid, pCAMBIA-1301 [con-ferring $\beta$-glucuronidase(GUS) activity and resistance to hygromycin] and cultured on selection medium (regeneration medium supplemented with hygromycin) to select putatively transformed shoots. The shoots were then rooted on root induction medium and transferred to pots containing sand and soil mixture in the ratio of 1:1. About 22 putative TO transgenic plants have been produced. Stable expression and integration of the transgenes in the putative transgenics were confirmed by GUS assay, PCR and Southern blot hybridization with a transformation efficiency of over 45%. Stable integration and expression of the marker gene has been confirmed in the TO and T1 transgenics through PCR, and Southern hybridization.