• Journal of Plant Biotechnology
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• v.38 no.4
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• pp.272-277
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• 2011

Plants expressing Agrobacterium sp. strain CP4 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) are known to be resistant to glyphosate, a potent herbicide that inhibits the activity of the endogenous plant EPSPS. In order to develop herbicide-resistant rice, we prepared transgenic rice plants with CP4 EPSPS gene under the control of CaMV 35S promoter for over-expression. A recombinant plasmid was transformed into rice via Agrobacterium-mediated transformation. A large number of transgenic rice plants were obtained with glyphosate and most of the transformants showed fertile. The integration and expression of CP4 EPSPS gene from regenerated plants was analyzed by Southern and northern blot analysis. The transgenic rice plants had CP4 EPSPS enzyme activity levels more than 15-fold higher than the wild-type plants. EPSPS enzyme activity of transgenic rice plants was also identified by strip-test method. Field trial of transgenic rice plants further confirmed that they can be selectively survived at 100% by spay of glyphosate (Roundup$^{(R)}$) at a regular dose used for conventional rice weed control.

• The Plant Pathology Journal
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• v.35 no.6
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• pp.575-584
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• 2019

Colletotrichum acutatum is a species complex responsible for anthracnose disease in a wide range of host plants. Strain C. acutatum KC05, which was previously isolated from an infected pepper in Gangwon Province of South Korea, was reidentified as C. scovillei using combined sequence analyses of multiple genes. As a prerequisite for understanding the pathogenic development of the pepper anthracnose pathogen, we optimized the transformation system of C. scovillei KC05. Protoplast generation from young hyphae of KC05 was optimal in an enzymatic digestion using a combined treatment of 2% lysing enzyme and 0.8% driselase in 1 M NH₄Cl for 3 h incubation. Prolonged incubation for more than 3 h decreased protoplast yields. Protoplast growth of KC05 was completely inhibited for 4 days on regeneration media containing 200 ㎍/ml hygromycin B, indicating the viability of this antibiotic as a selection marker. To evaluate transformation efficiency, we tested polyethylene glycol-mediated protoplast transformation of KC05 using 19 different loci found throughout 10 (of 27) scaffolds, covering approximately 84.1% of the entire genome. PCR screening showed that the average transformation efficiency was about 17.1% per 100 colonies. Southern blot analyses revealed that at least one transformant per locus had single copy integration of PCR-screened positive transformants. Our results provide valuable information for a functional genomics approach to the pepper anthracnose pathogen C. scovillei.

• Korean journal of applied entomology
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• v.35 no.1
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• pp.66-73
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• 1996

For the effective control of mosquito larvae, Anopheles sinensis, the expression vector pCYASK5-1 containing cryIVD gene of Bacillus thuringiensis subsp. morrisoni PG-14 was constructed and transformed into the cyanobacterium Synechocystis PCC6803. The transformants were selected on BG-11 medium containing kanamycin. The expression of cryIVD gene in transformant was confirmed by SDS-PAGE and Western blot analysis. The mortality of A. sinensis larvae was scored for 3 days. Furthermore, growth and distribution rate of transformant were examined. The results showed that Synechocystis PCC6803 transformed with cryIVD gene of B. thuringiensis subsp. morrisoni PG-14 was highly toxic to A. sinensis larvae, demonstrating that it will be a potential agent for mosquito control.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.49 no.1
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• pp.61-68
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• 2004

A reproducible transformation system via optimized regeneration media for Korean rice cultivars was established using Agrobacterium tumefeciens LBA4404 (pSBM-PPGN; gusA and bar). Although japonica rice genotypes were easier to produce transgenic plants compared to Tongil type cultivars, transformation efficiencies were not always correlated with regeneration efficiencies of non-transgenic callus on the control medium. Regeneration efficiencies of Donganbyeo, Ilmibyeo, and Manchubyeo were over 50% in non-transgenic control, however, transformation efficiencies were significantly low when only sucrose was added to the media as a carbon source. However, the medium, MSRK5SS-Pr (or MSRK5SM-Pr), that contains $5\textrm{mgL}^{-1}$ kinetin, $0.5\textrm{mgL}^{-1}$ NAA, 2 % sucrose (or maltose), 3% sorbitol, and $500\textrm{mgL}^{-1}$ proline, was the most efficient not only for regeneration of non-transgenic callus but also for regeneration of transgenic callus in the presence of L-phosphinotricin (PPT). Average transformation efficiencies of 16 Korean rice cultivars were significantly enhanced by using the optimized medium from 1.5% to 5.8% in independent callus lines and from 2.9% to 19.4% in tromsgenic plants obained. Approximately 98.9% (876 out of 885) transgenic plants obtained on optimized media showed basta resistance. Stable integration, inheritance and expression of gusA and bar genes were continued by GUS assay and PCR and Southern analysis of the bar gene. With Pst1 digestion of genomic DNA of transgenic plants, one to five copies of T-DNA segment were observed; however, 76% (19 out of 25 transgenic plants) has low copy number of T-DNA. The transformants obtained from one callus line showed the same copy numbers with the same fractionized band patterns.

• The Plant Pathology Journal
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• v.19 no.3
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• pp.162-165
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• 2003

Transformation of Fuji apple (Malus domestica 'Fuji') was performed using Agrobacterium tumefaciens harboring a coat protein (CP) gene of Cucumber mosaic virus (CMV). A plasmid DNA containing the virus CP and NPT II genes was introduced into the loaves of apple by th e Agrobacterium - mediated transformation procedure. Regenerated transformants of the apple were obtained by kanamycin resistance conferred by the introduced NPT II gene. PCR analysis showed that 3 out of 20 putatively selected R0 plant lines contain the CMV-CP gene. Nine putative transgenic lines out of 20 lines were investigated with the PCR analysis; 5 regenerants produced a 450 bp DNA band and 3 regenerants showed a 671 bp DNA band for the NPT II and CMV-CP genes, respectively. Southern hybyidization results demonstrate the successful integration of the CMV-CP gene into the genome of the apple. This is the first report on the generation of useful vius resistance source of transgenic apple for molecular breeding program.

• Journal of Plant Biotechnology
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• v.43 no.2
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• pp.240-247
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• 2016

In this study, routinely used transformation method, which includes transferring explants onto co-cultivation medium after inoculating them with bacterial solution for a while, was compared with 3 different inoculation methods. In every 3 methods, hypocotyl explants excised from 7-day-old sterile flax seedlings having cotyledon leaves and no root system dried under air flow in sterile cabin for 35 min were inoculated with different volumes of bacterial solution at different inoculation periods. GV2260 line of Agrobacterium tumefaciens having 'pBIN 19' plasmid containing npt II (neomycin phosphotransferase II) gene and GUS reporter gene was used in transformation studies. After inoculation, hypocotyl segments of seedlings (0.5 cm in length) - were excised and left to co-cultivation for 2 days. Then, explants were transferred to regeneration medium supplemented with different antibiotics. The presence of npt-II and GUS genes in transformants was confirmed by PCR and GUS analysis. The highest results in all characters examined in all cultivars were obtained from the 2 inoculation method in which hypocotyls excised from seedlings inoculated with $500{\mu}l$ of bacterial solution after drying in sterile cabin for 35 min were used.

• Journal of Plant Biotechnology
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• v.34 no.2
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• pp.145-152
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• 2007

Lactoferrin is an 80-kDa iron-binding glycoprotein known to exert many biological activities, such as facilitating iron absorption and having antimicrobial and anti-inflammatory effects. Rice can be a useful target for edible food plants to introduce human lactoferrin, because it has lower allergenicity and is likely to be safer than microorganisms or transgenic animals. A cDNA fragment encoding human lactoferrin (HLF) driven by the maize polyubiquitin promoter, along with herbicide resistance gene (bar) driven by CaMV 35S promoter, was introduced into rice (Oryza sativa L. cv. Dong Jin) using the Agrobacterium -mediated transformation system. Putative transformants were initially selected on the medium containing bialaphos. The stable integration of the bar and HLF genes into transgenic rice plants was further confirmed through polymerase chain reaction (PCR) and Southern blot analyses. The expression of the full length HLF protein from various tissues such as grains and young leaves of transgenic rice was verified by Western blot analysis. Analysis of progeny also demonstrated that introduced genes were stably inherited to the next generation at the Mendelian fashion.

• KSBB Journal
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• v.7 no.3
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• pp.191-200
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• 1992

Several strains of Agrobacterium tumefaciens were isolated from soil in the Taegu area and characterized to develop some useful vector systems for higher plant genetic engineering. The selected colonies had a unique form, and strains from the colonies were capable of tumor formation on the sunflower leaf surface. They had a large plasmid. The restriction analysis showed that they were another kinds of Ti plasmic compared with C58 and Ach5. The isolated strains were identified as the nopaline type and also as biovar 1 A. tumefaciens, according to their tumor morphology, blophyslcal and biochemical characteristics. One of the isolated strains, AK204 was transformed with binary vector (pGA642), having selectable marker (Kmr, Tcr). Furthermore, maize tissue cells were transformed by cocultivation with AK204/pGA642, and the transformants were selected on the selective medium and identified using PAGE patterns of their soluble proteins.

• KSBB Journal
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• v.11 no.1
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• pp.37-45
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• 1996

Marine bacterial strain, highly effective agar degrading, was isolated from south sea of Korea and was identified as Pseudomonas sp. This strain was named Halophilic Pseudomonas sp. W7 and accumulated an extracellular agarase which showed a high level of enzyme activity in the presence of agar and agarose. This extracellular agarase was purified by anion-exchange chromatography and gel filtration. Purified agarase showed a single protein band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its molecular weight was estimated to be about 89KDa. The agarase gene was cloned into Escherichia coli JM83 using the plasmid vector pUC19. DNA fragments(3.7, 3.0Kb) of Hind III-digested chromosomal DNA of Pseudomonas sp. W7 was inserted into the Hind III site of pUC19. Selected transformants, E. coli JM83/pSWl 000000and E. coli JM83/pSW3, produced agarase and this agarase was accumulated In the cytoplasmic space.

• BMB Reports
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• v.36 no.6
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• pp.586-592
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• 2003

Four putative promoters of the temperate mycobacteriophage L1 were cloned by detecting the $\beta$-galactosidase reporter expression in E. coli transformants that carried L1 specific operon-fusion library. All of the four L1 promoters were also found to express differentially in the homologous environment of mycobacteria. Of the four promoters, two were suggested to be the putative early promoters of L1 since they express within 0 to 10 min of the initiation of the lytic growth of L1. One of the putative early promoters showed a relatively better and almost identical activity in both E. coli and M. smegmatis. By a sequence analysis, we suggest that the L1 insert that contained the stronger early promoter possibly carries two convergent E. coli $\sigma^{70}$-like L1 promoters, which are separated from each other by about 300 nucleotides. One of them is the early promoter of L1 as it showed a 100% similarity with the early $P_{left}$ promoter of the homoimmune phage L5. The second promoter, designated P4, was suggested for its appreciable level of reporter activity in the absence of the -10 element of the $P_{left}$ equivalent of L1. By analyzing most of the best characterized mycobacteriophages-specific promoters, including the L1 promoter P4, we suggest that both the -10 and -35 hexamers of the mycobacteriophage promoters are highly conserved and almost similar to the consensus -10 and -35 hexamers of the E. coli $\sigma^{70}$ promoters.