• Korean Journal of Plant Tissue Culture
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• v.26 no.3
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• pp.163-169
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• 1999

This study was conducted to produce herbicide resistant plants by transferring phosphinothricin acetyltransferase (PAT) gene into Populus alba $\times$ Populus glandulosa No .3 using Agrobacterium tumefaciens MP 90/PAT. Leaf segments from in vitro grown shoots of hybrid poplar No. 3 were soaked in a AB medium containing Agrobacterium tumefaciens MP 90/PAT for 10 min and cocultivated for 2 days on MS medium containing 1.0 mg/L 2,4-D and 0.2mg/L kinetin (CIM). Putative transformed calli could be selected after cocultivation of leaf segments on CIM supplemented with 50mg/L kanamycin and 500mg/L cefotaxime for 3 weeks. The selected calli were cultured on CIM supplemented with 50 mg/L kanamycin and 500 mg/L cefotaxime for 5~8 weeks before transfer to WPM containing 1.0mg/L zeatin, 0.1mg/L BAP, 50 mg/L kanamycin and 500mg/L cefotaxime for shoot regeneration. Shoots were regenerated from the callus after 4 week cultivation, and the regenerants were grown on the same medium for 7~l0 weeks. The plants rooted on 1/2 WPM containing 0.2 mg/L IBA and 50 mg/L kanamycin. To confirm the gene insertion into plants, GUS activity was detected by histochemical assay in the transformed plants. Finally, the presence of both NPT II and PAT genes from the transgenic plants were confirmed by PCR amplification with the gene specific primers and subsequent PCR-Southern blot with DIG-labeled PAT gene probe. After acclimatization in pots for 4 weeks, the plants were sprayed by 3 mL/L of Basta to test resistance to the herbicide. The transgenic plants remained green, whereas all the control plants died after one week.

• Korean Journal of Weed Science
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• v.30 no.4
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• pp.380-389
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• 2010

Transgenic herbicide-resistant sweet potato plants [Ipomoea batatas (L.) Lam.] produced through a biolistic transformation were used in this study. The objective of this research was to find out a rapid and reliable assay method for confirming glufosinate-ammonium resistance. The techniques tested include whole-plant bioassay, one leaf bioassay, and leaf disk bioassay. Parameters investigated in this study were leaf injury and ammonium accumulation at 1 and 5 days after treatment of glufosinate-ammonium. In the leaf disk bioassay, leaf injury of the transgenic line 7171 was 1.9-fold less affected by glufosinate-ammonium than the wild type. The leaf injury of 7171 in one leaf and whole-plant bioassays was 59- and 92-fold less affected by glufosinate-ammonium, respectively, compared with that of the wild type. Leaf disk, one leaf, and whole-plant bioassays showed that ammonium accumulation of the 7171 was 2 to 20-, 4 to 43-, and 6 to 115-fold less affected by 0.5-5 mM glufosinate-ammonium than that of the wild type. All three bioassays successfully distinguished the resistance from the transgenic lines, but one leaf bioassay is the simplest and quickest. Leaf injury and ammonium accumulation were the same in leaves 1, 3, 5, 7, and 10 of 3 mM glufosinate-ammonium treated plants or nontreated plants. The one leaf bioassay was chosen as the standard procedure for future confirmation of resistance in transgenic sweet potato because it is a rapid and reliable assay.

• Korean Journal of Weed Science
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• v.30 no.2
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• pp.122-134
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• 2010

We investigated the levels of resistance and accumulation of terapyrroles, reactive oxygen species, lipid peroxidation, and antioxidative enzymes for reasons of growth reduction in herbicide-transgenic rice overexpressing Myxococcus xanthus, Arabidopsis thaliana, and human protoporphyrinogen oxidase (Protox) genes. The transgenic rice overexpressing M. xanthus (MX, MX1, PX), A. thaliana (AP31, AP36, AP37), and human (H45, H48, H49) Protox genes showed 43~65, 41~72 and 17~70-fold more resistance to oxyfluorfen, respectively, than the wild type. Among transgenic rice lines overexpressing Protox genes, several lines showed normal growth compared with the wild type, but several lines showed in reduction of plant height and shoot fresh weight under different light conditions. However, reduction of plant height of AP37 was much higher than other lines for the experimental period. On the other hand, the reduction of plant height and shoot fresh weight in the transgenic rice was higher in high light condition than in low light condition. Enhanced levels of Proto IX were observed in transgenic lines AP31, AP37, and H48 at 7 days after seeding (DAS) and transgenic lines PX, AP37, and H48 at 14 DAS relative to wild type. There were no differences in Mg-Proto IX of transgenic lines except for H41 and H48 and Mg-Proto IX monomethyl ester of transgenic lines except for MX, MX1, and PX. Although accumulation of tetrapyrrole intermediates was observed in transgenic lines, their tetrapyrrole accumulation levels were not enough to inhibit growth of transgenic rice. There were no differences in reactive oxygen species, MDA, ALA synthesizing capacity, and chlorophyll between transgenic lines and wild type indicating that accumulated tetrapyrrole intermediate were apparently not high enough to inhibit growth of transgenic rice. Therefore, the growth reduction in certain transgenic lines may not be caused by a single factor such as Proto IX, but by interaction of many other factors.

• Journal of Plant Biotechnology
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• v.30 no.1
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• pp.1-6
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• 2003

In an attempt to evaluate the current research status of genetically modified (GM) plants, the scientific research publications in Korea as well as in international SCI journals were screened. About 190 research articles related to the development of GM plants were searched from 10 different domestic journals in the last 12 years (Jan. 1990 to Sept. 2002), The researches in 65 articles were carried out with tobacco plant, 20 with rice, 19 with potatoes, and less then 9 articles from each other plant species, respectively, In total, 38 different plant species were being subjected for the development of GM plants. In particular, there was only one article for each major staple grains such as wheat, barley, soybean, and maize. In more than 47% of total published articles, scientists mainly focused on the basic research such as developing transformation system (46 articles), gene expression study in transgenic plants (34), and vector constructions (10). In addition, 28 articles which main authors are Korean scientists were searched from 11 different international SCI journals. Again, major plants for GM research were tobacco (10) and rice (7). More than 50% of published articles were focused on the basic research, gene expression study with transgenic plants (16). The publications on the research of disease-resistant plants were 7 articles, 3 for the development of stress-resistant and 2 for the herbicide-resistant plants, respectively. It is believed that the last 10 year's investment through government organizations has just strengthen the capacity for the next big stride on agricultural biotechnology in Korea.

• Journal of Life Science
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• v.19 no.7
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• pp.839-844
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• 2009

This experiment was conducted to evaluate pollen viability of Nakdongbyeo, transgenic rice lines, an F$_1$ hybrid from a cross between Milyang weedy rice and ABC-promoter transgenic rice line containing basta-resistant (bar) gene and subsequent selfed progenies, F$_2$ and F$_3$. The reaction of pollen with 3-{4,5 dimethylthiazolyl-2}-2,5-diphenyl monotetrazolium bromide (MTT) as a staining chemical immediately after pollen shedding showed maximum pollen viability of 86% in Nakdongbeyo, 75% in ABC-promoter transgenic rice line, 62% in ubiquitin-promoter transgenic line, 68% in F$_1$, 79% in F$_2$ and 78% in F$_3$. Viability gradually declined during subsequent observations at 20-minute intervals. However, there was a drastic decline in pollen viability after 40 minutes of pollen shedding. The mean difference of pollen viability among rice lines and time was highly significant, indicating significantly different pollen viabilities at different time intervals. Maximum viability of 36.2% was observed in F$_3$ and minimum viability of 3.5% was found in F$_2$ at 90 min after pollen shedding. Results of this experiment on pollen viability and longevity elucidate potential risks of pollen-mediated flow of herbicide-resistant gene from transgenic rice lines and possible integration of it into the weedy rice population.

• Korean Journal of Weed Science
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• v.18 no.3
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• pp.237-245
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• 1998

The transgenic tobacco (Nicotiana tabacum cv. Xanthi) plants containing Bacillus subtilis protoporphyrinogen oxidase gene with cauliflower mosaic virus 35S promoter have recently been generated by using Agrobacterium-mediated gene transformation. The nontransgenic and the transgenic tobacco plants were compared with respect to responses to diphenyl ether herbicide oxyfluorfen and under various environmental conditions. Both cellular leakage and lipid peroxidation caused by oxyfluorfen were found to be less in the transgenic than in the nontransgenic plants. Growth responses of the transgenic plants under various temperature, light, and water conditions were almost the same as those of the nontransgenic plants, although the transgenic plants exhibited slightly more retarded growth under low light or saturated water condition. These results revealed that the transgenic tobacco plants containing B. subtilis protoporphyrinogen oxidase gene under cauliflower mosaic virus 35S promoter were relatively resistant to oxyfluorfen and exhibited normal growth pattern. Possible mechanism of resistance to oxyfluorfen in the transgenic plants is also discussed.

• Proceedings of the Korean Society of Sericultural Science Conference
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• 1997.06a
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• pp.23-49
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• 1997

This is a progress report of rice biotechnology including development of gene transformation system, gene cloning and molecular mapping in rice. The scope of the research was focused on the connection between conventional breeding and biotech-researches. Plant transformation via Agrobacterium or particle bombardment was developed to introduce one or several genes to recommended rice cultivars. Two chimeric genes containing a maize ribosome inactivating protein gene (RIP) and a gerbicide resistant gene (bar) were introduced to Nipponbare, a Japonica cultivar, and transmitted to Korean cultivars. The homozygous progenies of herbicide resistant transgenic plant showed good fertility and agronomic characters. To explore the genetic resourses in rice, over 8,000 cDNA clones from immature rice seed have been isolated and sequenced. About 13% of clones were identified as enzymes related to metabolic pathway. Among them, twenty clones have high homology with genes encoding enzymes in the photorespiratory carbon cycle reaction. Up to now about 100 clones were fully sequenced and registered at EMBL and GenBank. For the mapping of quantitative tarits loci (QTL) and eternal recombinant inbred population with 164 F13 lines (MGRI) was developed from a cross between Milyang 23 and Gihobyeo, Korean rice cultivars. After construction of fully saturated RFLP and AFLP map, quantitative traits using MGRI population were analyzed and integrated into the molecular map. Eighty seven loci were determined with 27 QTL characters including yield and yield components on rice chromosomes. Map based cloning was also tried to isolate semi-dwarf (sd-1) gene in rice. A DNA probe, RG 109, the most tightly linked to sd-1 gene was used to screen from bacterial artifical chromosome (BAC) libraries and five over lapping clones presumably containing sd-1 gene were isolated. Rice genetic database including results of biotech reasearch and classical genetics is provided at Korea Rice Genome Server which is accessible with world wide web (www) browser. The server provides rice cDNA sequences and map informations linked with phenotypic images.

• Journal of Plant Biotechnology
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• v.38 no.4
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• pp.293-300
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• 2011

This study is conducted to develop an efficient transformation system via particle bombardment with PLBs (Protocorm-like bodies) in Cymbidium. For this, pCAMBIA3301 vector which carries a herbicide-resistant bar gene and gus gene as a reporter gene was used for transformation with Cymbidium cultivars 'Youngflower ${\times}$ masako' line. To select transformants, proper concentration of herbicide, PPT (phosphinotricin), should be determined. As a result, 5 mg/l of PPT was selected as a proper concentration. Further, proper conditions for particle bombardment were determined to obtain a high frequency of transformation. Results showed that 1.0 ${\mu}g$ of DNA concentration, 1,100 and 1,350 psi for helium gas pressure, 1.0 ${\mu}m$ of gold particle and 6 cm of target distance showed the best result for the particle bombardment experiment. Also, pre-treatment with combination 0.2 M sorbitol and 0.2 M mannitol for 4 hrs prior to genetic transformation increased the transformation efficiency up to 2.5 times. Using transformation system developed in this study, 3.2 ~ 4.0 transgenic cymbidium plants can be produced from 100 bombarded PLBs on average. Putative transgenic plants produced in this system confirmed the presence of the bar gene by PCR analysis. Also, leaves from randomely selected five transgenic lines were applied for Basta solution (0.5% v/v) to check the resistance to the PPT herbicide. As a result, three of them showed resistance and one of them showed the strongest resistance with the maintenance of green color as non-transformed plants showed. Using this established transformation system, more genes of interests can be introduced into Cymbidium plants by genetic transformation in the future.

• Journal of Plant Biotechnology
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• v.41 no.4
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• pp.216-222
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• 2014

This study was carried out to develop an efficient transformation protocol via particle bombardment with PLBs (protocorm-like bodies) in Phalaenopsis. To achieve this aim, osmoticum treatment and an increasing shooting chances in particle bombardment process were applied for this study. In addition, pCAMBIA3301: ORE7 vector which contains a herbicide-resistance bar gene as a selectable marker and ORE7 gene as a gene of interests were employed. With regard to the increasing chances of shooting in particle bombardment, double shooting was the best results with 1.5 ~ 2.5 times higher than those of a single or triple shooting treatment in the productioon of PPT (D-L-phosphinothricin)-resistant PLBs. However, regeneration rate of shoots in double shooting was not high as a single shooting. Further, double shooting showed 35 ~ 40% higher than that of a single shooting in the frequency of browning. Regarding effects of different osmotic treatments, combination of 0.2 M sorbitol with 0.2 M mannitol showed the best results in transformation efficiency, regeneration of transformants and reduction of browning. Putative transgenic Phalaenopsis plants were analyzed by PCR analysis and confirmed the presence of bar and ORE 7 gene. Also, real-time PCR was conducted by using 21 transgenic plants and showed only 4 plants had one copy of transgene; whereas, the other 17 plants had more than 2 copies of transgene. Transgenic phalaenopsis plants produced in this study were transferred to pots and flowered normally without morphological variations in flower and leaf.

• Korean Journal of Weed Science
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• v.18 no.3
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• pp.205-213
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• 1998

This experiment was carried out to produce herbicide resistant potatoes hawing only chimeric phosphinothricin acetyltransferase (PAT) genes without using antibiotic selectable marker. The pDY502 vector having only PAT gene was reconstructed for transformation of potato. The reconstructed vector was introduced to Agrobacterium tumefaciens MP90 disarmed, and they were used for potato transformation. Hormonal requirement for plant regeneration from leaves and stem explants of potato was investigated. From this experiment, MS medium treated with IBA 0.1 mg/L + BA 0.5 mg/L was the best for potato regeneration, and the ratio of shoot regeneration was 54% for leaf and 46% for stem in that condition. For transformation, explants of potato leaves and stems were cocultured with A. tumefaciens MP90 containing reconstructed vector harvoring only PAT gene. When the potato explants were placed on various concentrations of bialaphos and all the potato explants were dead on medium with over 5.0mg/L bialaphos. By this selection methods, the explants cocultured with Agrobacterium produced the putative transgenic shoots on medium with 5mg/L bialaphos treatment after 3-4 weeks. Second selection was performed by transferring the shoot tips of putative transgenic to medium containing 20mg/L of bialaphos. The shoot tips grew well on the second selection medium, indicating the production of successful transgenic plants. But normal shoots were dead in same cytotoxic medium. Incorporation of the PAT gene into transgenic potatos were confirmed by PCR analysis of DNA and Southern hybridization. These results show that the PAT gene can serve as a selectable marker and herbicide resistant genes for transformation of potato.