• Journal of Plant Biotechnology
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• v.38 no.3
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• pp.198-202
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• 2011

Agrobacterium tumefaciens-mediated cotyledonary-node explants transformation was used to produce transgenic cucumber. Cotyledonary-node explants of cucumber (Cucumis sativus L. cv., Eunsung) were co-cultivated with Agrobacterium strains (EHA101) containing the binary vector (pPZP211) carrying with CaMV 35S promoter-nptII gene as selectable marker gene and 35S promoter-DQ gene (unpublished data) as target gene. The average of transformation efficiency (4.01%) was obtained from three times experiments and the maximum efficiency was shown at 5.97%. A total of 9 putative transgenic plants resistant to paromomycin were produced from the cultures of cotyledonary-node explants on selection medium. Among them, 6 transgenic plants showed that the nptII gene integrated into each genome of cucumber by Southern blot analysis.

• Journal of Microbiology and Biotechnology
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• v.25 no.7
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• pp.988-998
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• 2015

The filamentous fungus Aspergillus oryzae is a well-known expression host used to express homologous and heterologous proteins in a number of industrial applications. To facilitate higher yields of proteins of interest, we constructed the pAsOP vector to express heterologous proteins in A. oryzae. pAsOP carries a selectable marker, pyrG, derived from Aspergillus nidulans, and a strong promoter and a terminator of the amyB gene derived from A. oryzae. pAsOP transformed A. oryzae efficiently via the PEG-CaCl₂-mediated transformation method. As proof of concept, green fluorescent protein (GFP) was successfully expressed in A. oryzae transformed by pAsOP-GFP. Additionally, we identified a novel fungal α-amylase (PcAmy) gene from Penicillium sp. and cloned the gene into the vector. After transformation by pAsOPPcAmy, the α-amylase PcAmy from Penicillium sp. was successfully expressed in a heterologous host system for the first time. The α-amylase activity in the A. oryzae transformant was increased by 62.3% compared with the untransformed A. oryzae control. The PcAmy protein produced in the system had an optimum pH of 5.0 and optimum temperature of 30^oC. As a cold-adapted enzyme, PcAmy shows potential value in industrial applications because of its high catalytic activity at low temperature. Furthermore, the expression vector reported in this study provides promising utility for further scientific research and biotechnological applications.

• Korean Journal of Agricultural Science
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• v.47 no.4
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• pp.815-827
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• 2020

The epidermal growth factor (EGF) transgenic soybean was developed and biosynthesis of human epidermal growth factor (hEGF) in soybean seeds was confirmed. Also, EGF transgenic soybean were found to contain a herbicide resistance selectable marker by introduction of phosphinothricin acetyltransferase (PAT) gene from the Streptomyces hygroscopicus. For biosafety assessment, the EGF transgenic soybean expressing the EGF biosynthesis gene EGF and herbicide resistant gene PAT was tested to determine effects on survival of Cyprinus carpio, commonly used as a model organism in ecotoxicological studies. C. carpio was fed 100% ground soybean suspension, EGF soybean or non-genetically modified (GM) counterpart soybean (Gwangan). Gene expression of EGF soybean was confirmed by PCR and ELISA to have EGF/PAT. Feeding test showed that no significant differences in cumulative immobility or abnormal response between C. carpio samples fed on EGF soybean and non-GM counterpart soybean. The 48 h-EC50 values of the EGF and non-GM soybean were 1,688 mg·L-1 (95% confidence limits: 1,585 - 1,798 mg·L-1) and 1,575 mg·L-1 (95% confidence limits: 1,433 - 1,731 mg·L-1), respectively. The soybean NOEC (no observed effect concentration) value for C. carpio was suggested to be 625 mg·L-1. We concluded that there was no significant difference in toxicity for non-target organisms (C. carpio) between the EGF soybean and non-GM counterparts.

• Korean Journal of Breeding Science
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• v.42 no.5
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• pp.507-514
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• 2010

Peanut(Arachis hypogaea L.) is one of the major oilseed crops. The peanut oil consists of palmitic, oleic and linoleic acids, which are present at levels of 10%, 36-67% and 15-43%, respectively. High oleate mutant of peanut F435 contains 80% oleate and as little as 2% linoleate in seed oil. Previous study indicated that delta 12 fatty acid desaturase is a major enzyme controlling the oleate content in seeds of oilseed crops. F435 sequence alignment of their coding regions disclosed that an extra A(adenine) was inserted at the position +2,823 bp of delta 12 fatty acid desaturase gene. This study was to develop molecular marker (SNP marker) co-segregating with the high oleate trait. Chopyeong ${\times}$ F435 $F_2$ 41 population were investigated using molecular marker and fatty acid assay (NIR and gas chromatography). Finally, this marker segregates Chopyeong type 26 lines, heterotype 9 lines and F435 type 6 lines. These results in our study suggested that SNP marker conform fatty acid assay.

• Journal of Microbiology and Biotechnology
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• v.25 no.7
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• pp.1015-1025
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• 2015

The development of diverse polymyxin derivatives is needed to solve the toxicity and resistance problems of polymyxins. However, no platform has generated polymyxin derivatives by genetically engineering a polymyxin synthetase, which is a nonribosomal peptide synthetase. In this study, we present a two-step approach for the construction of engineered polymyxin synthetases by substituting the adenylation (A) domains of polymyxin A synthetase, which is encoded by the pmxABCDE gene cluster of Paenibacillus polymyxa E681. First, the seventh L-threonine-specific A-domain region in pmxA was substituted with the L-leucine-specific A-domain region obtained from P. polymyxa ATCC21830 to make polymyxin E synthetase, and then the sixth D-leucine-specific A-domain region (A_6-D-Leu-domain) was substituted with the D-phenylalanine-specific A-domain region (A_6-D-Phe-domain) obtained from P. polymyxa F4 to make polymyxin B synthetase. This step was performed in Escherichia coli on a pmxA-containing fosmid, using the lambda Red recombination system and the sacB gene as a counter-selectable marker. Next, the modified pmxA gene was fused to pmxBCDE on the chromosome of Bacillus subtilis BSK4dA, and the resulting recombinant strains BSK4-PB and BSK4-PE were confirmed to produce polymyxins B and E, respectively. We also succeeded in constructing the B. subtilis BSK4-PP strain, which produces polymyxin P, by singly substituting the A_6-D-Leu-domain with the A_6-D-Phe-domain. This is the first report in which polymyxin derivatives were generated by genetically engineering polymyxin synthetases. The two recombinant B. subtilis strains will be useful for improving the commercial production of polymyxins B and E, and they will facilitate the generation of novel polymyxin derivatives.

• Journal of Korean Society of Forest Science
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• v.86 no.4
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• pp.407-414
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• 1997

The resistance of a non-transgenic poplar clone, 'Ogy' and three transgenic poplar lines to the cottonwood leaf beetle, Chrysomela scripta F., was evaluated by in vitro feeding. The lines were transformed with neomycin phosphotransferase II(NPT II) as a selectable marker, proteinase inhibitor II(pin2) as a resistance gene, and CaMV 35S as a promoter. An efficient method of sterilizing the beetle eggs and introducing them into plant tissue cultures was developed. The resistance of the transgenic lines was investigated in terms of effects tin leaf area consumed, insect weight, insect developmental stages, and plantlet root dry weight after feeding. Also, leaf area consumed was examined by leaf age as measured through leaf plastochron index(LPI). The leaf area consumed and insect weight were highly significant between transformants and control, and insect development in vitro was significant among the transgenic lines. Larval infestation was the most severe around LPI 4 to 5 which were young leaves. The system provided a quick, highly controlled method to screen developing transgenic plantlets directly.

• Journal of Plant Biotechnology
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• v.6 no.1
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• pp.25-37
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• 2004

Successful genetic transformation of plants requires non-chimeric selection of transformed tissues and their subsequent regeneration. With rare exceptions, most transformation protocols still rely heavily on antibiotics for selecting transgenic cells that contain an antibiotic-degrading selectable marker gene. Here, the morphogenic capacity of in-vitro explants of chrysanthemnum and tobacco stems and leaves (control and transgenic) changed with the addition of aminoglycoside antibiotics (AAs), In a test of 6 AAs, phytotoxicity occurred at concentrations of 10 to 25 and 50 to 100$\mu\textrm{g}$ $mL^{-1}$ in chrysanthemum and tobacco explants, respectively. Light conditions as well as explant source and size also had significant effects. The use of transverse thin cell layers (tTCLs), in conjunction with high initial AA selection levels, supported the greatest regeneration of transgenic material (adventitious shoots or callus) and the lowest number of escapes. Flow-cytometric analyses revealed no endodu-plication in chrysanthemum, even at high AA levels. However, this phenomenon was observed in tobacco calli(8C or more), even at low AA concentrations (i.e., 5 to 10 $\mu\textrm{g}$ mL$^{-1}$ ).

• Horticultural Science & Technology
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• v.19 no.1
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• pp.17-21
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• 2001

To develop a selection system for regenerating plants from transformed tissues, effects of four antibiotics (kanamycin, hygromycin, carbenicillin, cefotaxime) and herbicide (phosphinotricin) on shoot regeneration from cotyledon and hypocotyl explants of Chinese cabbage (Brassica campestris L. ssp. pekinensis) were studied. For cotyledon, shoot induction was not significantly affected by kanamycin at $1mg{\cdot}L^{-1}$, but the number of shoots formed was significantly reduced at $2mg{\cdot}L^{-1}$, and no shoots were regenerated from any explants at $6mg{\cdot}L^{-1}$ or higher. Hypocotyl explants showed similar result as cotyledon. Kanamycin at $7mg{\cdot}L^{-1}$ may be adequate for selecting Chinese cabbage transformants. Hygromycin at $4mg{\cdot}L^{-1}$ or higher completely inhibited the growth and shoot regeneration of Chinese cabbage explants. Therefore, resistance gene to hygromycin may also be used as a selective marker for Chinese cabbage transformation. Carbenicillin and cefotaxime, the cephalosporin type of antibiotics, had little effect on shoot regeneration of Chinese cabbage explants. Since carbenicillin and cefotaxime have low toxicity to Chinese cabbage, they are suitable for use in tissue culture to eliminate Agrobacterium in transformation experiments after co-cultivation. Shoot regeneration from cotyledon and hypocotyl explants was significantly reduced in presence of $1mg{\cdot}L^{-1}$ phosphinotricin (PPT) and completely inhibited by $2mg{\cdot}L^{-1}$ or higher. PPT, same as antibiotics, may also be used to select transformed cells. Since Chinese cabbage is known to be recalcitrant to in vitro shoot regeneration compared to other Brassica species, even though lower levels of selectable markers result in more transformants but simultaneously allow more untransformed escapes to develop, lower levels of antibiotics and herbicides could be successfully used as a selectable marker to reduce selection pressure.

• The Journal of Natural Sciences
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• v.4
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• pp.103-142
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• 1991

These studies were carried out to select somatic hybrid using selectable marker genes of Nicotiana glauca transformed by NPTII gene and Solanum tuberosum transformed by T- DNA, and to study characteristics of transformant. The results are summarized as follows. 1. Crown gall tumors and hairy roots were formed on potato tuber disc infected by A. tumefaciens Ach5 and A. rhizogenes ATCC15834. These tumors and roots could be grown on the phytohormone free media. 2. Callus formation from hairy root was prompted on the medium containing 2, 4 D 2mg/I with casein hydrolysate lg/l. 3. The survival ratio of crown gall tumor callus derived from potato increased on the medium containing the activated charcoal 0. 5-2. 0mg/I because of the preventions on the other hand, hairy roots were necrosis on the same medium. 4. Callus derived from hairy root were excellently grown for a short time by suspension culture on liquid medium containing 2, 4-D 2mg/I and casein hydrolysate lg/l. 5. The binary vector pGA643 was mobilized from E. coli MC1000 into wild type Agrobacteriurn tumefaciens Ach5, A. tumefaciens $A_4T$ and disarmed A. tuniefaciens LBA4404 using a triparental mating method with E. ccli HB1O1/pRK2013. Transconjugants were obtained on the minimal media containing tetracycline and kanamycin. pGA643 vectors were confirmed by electrophoresis on 0.7% agarose gel. 6. Kanamycin resistant calli were selected on the media supplemented with 2, 4-D 0.5mg/1 and kanamycin $100\mug$/ml after co- cultivating with tobacco stem explants and A. tumefaciens LBA4404/pGA643, and selected calli propagated on the same medium. 7. The multiple shoots were regenerated from kanamycin resistant calli on the MS medium containing BA 2mg/l. 8. Leaf segments of transformed shoot were able to grow vigorusly on the medium supplemented with high concentration of kanamycin $1000\mug$/ml. 9. Kanamycin resistant shoots were rooting and elongated on medium containing kanamycin $100\mug$/ml, but normal shoot were not. 10. For the production of protoplast from potato calli transformed by T-DNA and mesophyll tissue transformed by NPTII gene, the former was isolated in the enzyme mixture of 2.0% celluase Onozuka R-10, 1.0% dricelase, 1.0% macerozyme. and 0.5M mannitol, the latter was isolated in the enzyme mixture 1.0% Celluase Onozuka R-10, 0.3% macerozyme, and 0.7M mannitol. 11. The optimal concentrationn of mannitol in the enzyme mixture for high protoplast yield was 0.8M at both transformed tobacco mesophyll and potato callus. The viabilities of protoplast were shown above 90%, respectively. 12. Both tobacco mesophyll and potato callus protoplasts were fused by using PEG solution. Cell walls were regenerated on hormone free media supplemented with kanamycin after 5 days, and colonies were observed after 4 weeks culture.

• Journal of Plant Biology
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• v.35 no.3
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• pp.237-245
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• 1992

To understand the properties of the cauliflower mosaic virus (CaMV) 35S promoter and the effect of the deleted maize alcohol dehydrogenase I-S (Adhl-S) intron 1 on the expression of the CaMV $35S{\beta}-glucuronidase$ (GUS) gene in potato (Solanum tuberosum L. cv. Superior), we constructed a chimeric gene and transferred it into potato with Agrobacterium tumefaciens mediated method. The pLS201, a gene transfer vector of 17.7 kilobase pairs, was composed of the CaMV 35S promoter, the 249 base pairs of deleted maize Adhl-S intron 1, the GUS reporter gene, and the kanamycin resistance gene as a selectable marker for transformation. The GUS activity was examined by histochemical and spectrophotometric assay in transformed potato plants. The GUS activity was found primarily around the vascular tissue cells in stem and root. In the spectorophotometric assay, the level of GUS activity of transgenic potato transformed with CaMV 35S/249 bp of intron 1 fragment-GUS (pLS201) was compared with that of potato transformed with CaMV 35S-GUS (pBI121). The quantitative spectrophotometric assay showed that the level of GUS activity in potato transformed with pLS201 was higher in leaf, stem and root by 30-, 34- and 42-fold, respectively than those in potato transformed with pBI121. This results indicate that the inclusion of the deleted maize Adhl-S intron 1 resulted in increament of the GUS gene expression in transgenic potato.potato.