• Journal of Plant Biotechnology
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• v.32 no.2
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• pp.91-95
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• 2005

Agrobacterium tumefaciens-mediated immature embryo transformation was used to produce transgenic maize. Immature embryo of Hi II genotype were co-cultivated with strains Agrobacterium tumefaciens (C58C1) containing the binary vectors (pPTN290) carrying with Ubiquitin promoter-GUS gene as reporter gene and NOS promoter-nptll gene conferring resistance to paromomycin as selective agent. Seven embryogenic callus lines transformed showed the resistance in paromomycin antibiotics. Histochemical GUS assay showed that 7 individual lines transformed with the GUS gene were positive response among the transformants. Southern blot analysis revealed that the nptll gene segregated and expressed in their progeny.

• 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.

• 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.

• Journal of Korean Society of Forest Science
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• v.77 no.2
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• pp.199-207
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• 1988

Three clones of Populus alba ${\times}$ P. grandidentata have been tested for susceptibility to Agrobacterium tumefaciens strains A281 and A348. We determined the optimum concentration of kanamycin sulfate for effective selection of leaf disc-derived, transgenic tissues transformed using Agrobacterium binary vector pGA472 containing a neomycin phosphotransferase gene (NPT-II) which confers kanamycin resistance. Of the wild type Ti plasmids contained by the two Agrobacterium strains, pTiBo542 of strain A281 appears to be best suited to serve as a helper plasmid for binary vector systems. A relatively low concentration (10mg/l) of kanamycin sulfate inhibited adventitious shoot initiation from leaf discs on regeneration medium. Transformed kanamycin-resistant calli were obtained by culturing Agrobacterium inoculated leaf discs on selective regeneration medium. The transformed kanamycin-resistant calli continued to grow on regeneration media supplemented with kanamycin sulfate to levels of 50 and 200mg/l. The growth of non-co-cultivated control calli was severely inhibited on regeneration medium containing 50mg/l kanamycin sulfate.

• Journal of Plant Biotechnology
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• v.29 no.2
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• pp.93-98
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• 2002

A VP6 fragments was subcloned with BamHI in the binary pMBP-1 vector under Califlower Mosaic Virus (CaMV) 355 promoter and neomycin phosphotransferase II (npt II) gene. The recombinant binary vector was mobilized into Agrobacterium-tumefaciens LBA4404 by the freeze-thaw method and potato (Solanum tubensum L. cv Desiree) was transformed by modified leaf-disc cocultivation. Shoots were induced on MS medium with 0.01 mg/L NAA, 0.1 mg/L GA$_3$, 2.0 mg/L Zeatin, 100.0 mg/L kanamycin, 500.0 mg/L carbenicillin. In order to identify the copy number of VP6 into potato plant, total genomic DNA was isolated from transgenic potato and analysed by Southern blotting. Genomic DNA and total mRNA analysis demonstrated the incorporation of the foreign gene into the potato genome, as well as their transcription.

• Journal of Korean Society of Forest Science
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• v.79 no.3
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• pp.278-284
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• 1990

We have demonstrated expression of bacterial genes transferred into cells of Populus nigra ${\times}$ P. maximowiczii by A. tumefaciens strain 6044 (pGA 472). We determined the optimum concentration of kanamycin sulfate for effective selection of punctured leaf transformed using Agrobacterium binary vector pGA 472 containing a neomycine phosphotransferase gene (NPT-II) which confers kanamycin resistance. The combination of cefotaxime (200mg/l) and carbenicillin (300mg/l) showed good performance of discarding Agrobacterium from inoculated punctured leaf. A relatively low concentration (10mg/l) of kanamycin sulfate inhibited callus and shoots induction from punctured leaf. Number of shoots regenerated from co-cultured punctured leaf was 3.0 on MS basal medium supplemented with 10 mg/l kanamycin sulfate, while that of not co-cultured punctured leaf was none. The regeneration rate was 10% from the punctured leaf co-cultured on MS medium with 10 mg/l kanamycin. Regenerated shoots are developing from micropropagation for Southern blot analysis and inheritance of the kanamycin resistance trait (NPT-II).

• Korean Journal of Plant Resources
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• v.10 no.4
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• pp.300-304
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• 1997

Leaf dise explants of Solanum tuberosum cultivar. Desiree and Atlantic, were infected with a Agrobacterium MP90 strain containing chimeric gene construct, consisting of antibiotic and low temperature regulated gene (H28) for transformation. regenerated multiple shoots were selected on a medium containing kanamycin and carbenieillin after exposure to Agrobacterium. Both PCR analysis of NPT Ⅱ, H28 genes and northern blot analysis indicated that the genes coding for the enzyme were successfully integrated into the potato genome and could be expressed in potato plants.

• Korean Journal of Medicinal Crop Science
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• v.15 no.2
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• pp.95-99
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• 2007

The cost of conventional cultivation of ginseng (Panax ginseng C.A. Meyer) is very expensive, because shadow condition should be maintained during cultivation periods owing to inherently weak plant for high-temperature. Therefore, application of plant biotechnology may be possible to overcome these difficulties caused by conventional breeding of ginseng. Transgenic plants were produced via Agrobacterium tumefaciens Gv3101, both carrying the binary plasmid pBI121 mLPISO with nptII and Iso (isoprene synthase) gene. Integration of the transgenes into the P. ginseng nuclear genome was confirmed by PCR analysis using nptII primers and Iso primers. RT-PCR result also demonstrated the foreign isoprene synthase gene in three transgenic plant lines (T1, T3, and T5) which was expressed at the transcriptional level. When whole plants of transgenic ginseng were exposed to high temperature at $46^{\circ}C$ for 1 h, a non-transformed plant was wilted from heat shock, whereas a transgenic plant appeared to remain healthy. We suggest that the introduction of exogenous isoprene synthase is considered as alternative methods far generating thermotolerance ginseng.

• Plant Biotechnology Reports
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• v.4 no.4
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• pp.293-301
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• 2010

The feasibility of two strategies for transgene pyramiding using Agrobacterium-mediated transformation was investigated to develop a transgenic potato (Solanum tuberosum L. cv. Iwa) with resistance to potato tuber moth (PTM) (Phthorimaea operculella (Zeller)). In the first approach, cry1Ac9 and cry9Aa2 genes were introduced simultaneously using a kanamycin (nptII) selectable marker gene. The second approach involved the sequential introduction (re-transformation) of a cry1Ac9 gene, using a hygromycin resistance (hpt) selectable marker gene, into an existing line transgenic for a cry9Aa2 gene and a kanamycin resistance (nptII) selectable marker gene. Multiplex polymerase chain reaction (PCR) confirmed the presence of the specific selectable marker gene and both cry genes in all regenerated lines. The relative steady-state level of the cry gene transcripts in leaves was quantified in all regenerated lines by real-time PCR analysis. Re-transformation proved to be a flexible approach to effectively pyramid genes for PTM resistance in potato, since it allowed the second gene to be added to a line that was previously identified as having a high level of resistance. Larval growth of PTM was significantly inhibited on excised greenhouse-grown leaves in all transgenic lines, although no lines expressing both cry genes exhibited any greater resistance to PTM larvae over that previously observed for the individual genes. It is anticipated that these lines will permit more durable resistance by delaying the opportunities for PTM adaptation to the individual cry genes.

• Korean Journal of Plant Resources
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• v.17 no.2
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• pp.161-168
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• 2004

Optimal regeneration conditions of ginseng transformants were studied. It has been known that Ferritin Light Heavy Chain (FLHC) gene remove the several heavy metal by combination, store and transport. To obtain the ginseng tolerant to heavy metal, binary vector was introduced in Agrobacterium by tri-parental mating and then Agrobacterium tumefaciens MP90/FLHC was selected on the AB media and MS media containing kanamycin. Explants were co-cultured with Agrobacterium tumefaciens MP90/FLHC, which contained NPT II as a selectable marker, tadpole ferritin heavy chain (FLHC) gene and human ferritin light chain gene and then a number of embryos were induced. The induced embryo transferred to shooting media consisting of MS medium supplemented with GA 10 mg/L. As a result of examination that induced the normal growth of transfomants, transformants showed the equivalent growth in both root and shoot on the media containing the 1/3 MS.