• Journal of Plant Biotechnology
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• v.3 no.3
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• pp.113-121
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• 2001

The use of a marker gene in a transformation process aims to give a selective advantage to the transformed cells, allowing them to grow faster and better, and to kill the non-transformed cells. In general, the selective gene is introduced into plant genome along with the genes of interest. In some cases, the marker gene can be the gene of interest that will confer an agronomic characteristic, such as herbicide resistance. In this review we list and discuss the use of the most common selective marker genes on plant transformation and the effects of their respective selective agents. These genes could be divided in categories according their mode of action: genes that confer resistance to antibiotics and herbicides; and genes for positive selection. The contention of the marker gene flow through chloroplast transformation is further discussed. Moreover, strategies to recover marker-free transgenic plants, involving multi-auto-transformation (MAT), co-transformation, site specific recombination and intragenomic relocation of transgenes through transposable elements, are also reviewed.

• Korean Journal of Veterinary Research
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• v.24 no.1
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• pp.40-49
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• 1984

To investigate the factors influencing the artifical transformation in Escherichia coli, E. coli C600 was transformed by pBR322 DNA with tetracycline and ampicillin resistant gene purified by CsCl-Etbr equilibrium density gradient centrifugation from E.coli HB 101. The influencing factors in the transformation such as concentration of calcium chloride, time of ice incubation, temperature and time of heat shock, time of gene expression, effects of plasmid DNA concentration and adding time were examined in these experiments. The results obtained were as follows; 1. The highest transformation frequency was observed in the treatments of 100 mM $CaCl_2$ before heat shock and the treatment of $CaCl_2$ was essential step in the process of E. coli transformation. 2. The highest transformation frequency was observed in the treatment of heat shock at $42^{\circ}C$ for 4 min. or $37^{\circ}C$ for 6 min., but the prolonged heat shock resulted a decreased transformation frequency. 3. Treatments of ice incubation at $0^{\circ}C$ for 45 min. before heat stocks or at $0^{\circ}C$ for 30min. after heat shock resulted an increased transformation frequency. 4. There was a linear relationship between DNA concentration and transformation frequency at the concentration of $8{\times}10^3$ recipient cells. The highest transformation frequency reached in carte of 7 mcg of donor DNA, but above 1 mcg of DNA concentration, transformation frequency was not remarkably increased. Addition of donor DNA just after the treatment of $CaCl_2$ was the best. 5. The best condition of gene expression at $37^{\circ}C$ were 40min. for TC-resistant gene and 100min. for AP-resistant gene. TC-resistant gene was higher in the transformation frequency and faster in the gene expression time than AP-resistant gene. In these results, the best conditions for the transformation of E. coli C 600 with pBR322 DNA were: treatment with 100mM $CaCl_2$, ice incubation at $0^{\circ}C$ for 45 min, heat shock at $42^{\circ}C$ for 4 min., 30 min. of ice incubation and incubation at $37^{\circ}C$ for 100min. for gene expression in that order.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.49 no.5
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• pp.434-439
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• 2004

Medicago truncatula is a model plant for molecular genetic studies of legumes and plant-microbe interactions. To accelerate finding of genes that play roles in the early stages of nodulation and stress responses, a trans-genic plant was developed that contains a promoter­reporter fusion. The promoter of rip], a Rhizobium-induced peroxidase gene, was fused to the coding region of $\beta-glucuronidase (GUS)$ gene and inserted into a modified plant transformation vector, pSLJ525YN, in which the bar gene was preserved from the original plasmid but the neomycin phosphotransferase gene was replaced by a polylinker. Transformation of M. truncatula was carried out by vacuum infiltration of young seedlings with Agrobacterium. Despite low survival rates of infiltrated seedlings, three independent transformants were obtained from repeated experiments. Southern blot analyses revealed that 7 of 8 transgenic plants of the T 1 generation contained the bar gene whereas 6 $T_1$ plants contained the GUS gene. These results indicate that vacuum infiltration is an effective method for transformation of M. truncatula. The progeny seeds of the transgenic plants will be useful for mutagenesis and identification of genes that are placed upstream and may influence the expression of rip] in cellular signaling processes including nodulation.

• The Plant Pathology Journal
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• v.20 no.2
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• pp.158-163
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• 2004

Chlorella is a eukaryotic microalgae which shares metabolic pathways with higher plants. These charac-teristics make chlorella a potential candidate for eukaryotic overexpression systems. Recently, a foreign flounder growth hormone gene was stably introduced and expressed in transformed Chlorella ellipsoidea by using a modified plant transformation vector that contains cauliflower mosaic virus (CaMV) 35S pro-moter and the phleomycin resistant Sh ble gene as a selection marker. In this study, this same vector was modified by incorporating a promoter and a 3' UTR region of the 33kDa peptide gene from a chlorella virus that was isolated in our laboratory. The 33kDa gene promoter was used to replace the 35S promoter and the 3' UTR was introduced to separate the target gene and downstream Sh ble gene. Three different chlorella transformation vectors containing human erythropoietin (EPO) gene were constructed. The mp335EPO vector consists of a promoter from the 33kDa peptide gene, whereas the mp3353EPO vector contains the same promoter from the 33kDa peptide gene and its 3' UTR. The mp35S33pEPO vector contains the 35S promoter and the 3' UTR from the 33 kDa peptide gene. There was no significant difference in the expression levels of EPO protein in chlorella cells transformed with either of three of the transformation vectors. These data indicate that the promoters from the chlorella virus are comparable to the most common CaMV 35S promoter. Furthermore, these data suggest that other promoters from this virus can be used in future construction of chlorella transformation system for higher expression of target proteins.

• Journal of Crop Science and Biotechnology
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• v.11 no.4
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• pp.233-236
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• 2008

A positive selectable marker system was adapted for transformation of mature embryo-derived calli of Indica rice (Oryza sativa L.) utilizing the PMI gene encoding for phosphomannose-isomerase that converts mannose-6-phosphate to fructose-6-phosphate. The transformed cells grew on medium supplemented with 3% mannose as carbon source and calli were selected on media containing various concentrations of mannose. Molecular analyses showed that the transformed plants contained the PMI gene. The results indicate that the mannose selection system can be used for Agrobacterium-mediated transformation of mature embryo in rice to substitute the use of conventional selectable markers in genetic transformation.

• Korean Journal of Plant Tissue Culture
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• v.28 no.3
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• pp.147-151
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• 2001

Explants of Lactuce sativa cultivar, chungchima, were co-cultivated with Agrobacterium tumefaciences LBA4404, EHA101 strains containing nptll gene and ferritin gene encoding iron storage protein from soybean for transformation. Through initial selection of regenerated explants by culturing on a kanamycin and carbenicillin containing MS medium, multiple shoots were obtained after 2 months of culture. For a complementary step of selection, putative transgenic shoots were transferred to 1/2 MS basal medium supplemented with 100 mg/L kanamycin and 500 mg/L carbenicillin. The selected shoots were tested with PCR analysis using nptll, ferritin specific primers whether ferritin gene was introduced to genome of the plants. These results confirmed that produced the specific PCR bands in the putative transgenic lines. Additionally the Northern blot showed that transcripts of ferritin gene were detected in mature leaf of the transgenic lines. These results suggest that ferritin gene be successfully integrated and transcribed in the putative transgenic lettuce plants.

• Journal of The Korean Society of Grassland and Forage Science
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• v.20 no.1
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• pp.13-18
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• 2000

An efficient method for Agrobacterium-mediated transformation of forage crop alfalfa (Medicago sativa L.) was established using secondary somatic embryogenic calli. Agrobacterium tumefaciens strain EHAlOl and a binary vector pIG121-Hm which has selection markers for kanamycin and hygromycin have been shown to be an efticient materials for alfalfa transformation. The secondary somatic embryogenic calli originated from hypocotyl explants of alfalfa were efficient infection materials for Agrobacterium EHAlOl and normally germinated into plantlets. The introduced gene (GUS) was constitutively expressed in all tissues of transgenic alfalfa with different expression levels. These results indicate that the use of pIG121-Hm vector, Agrobacterium EHAlOl and improved culture system of callus facilitate the transformation of alfalfa. (Key words : Agrobacterium, Alfalfa, Gene transfer, Transformation)

• Journal of Life Science
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• v.16 no.3 s.76
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• pp.365-368
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• 2006

Yeast mitochondrial transformation has been confirmed by cell death and CFP expression (CDF: cell death factor gene). Expression vector containing CDF and CFP driven by one TPI (Triose-phosphate isomerase) promoter (called artificial operon type) was bombarded to Yeast. Interestingly, yeast cells were progressively deformed into unusual shapes and lysed inner cytoplasm resulting in ell death after all after bombarding with expression vector (CDC and GFP). Since there is no report about more than one gene expression simultaneously in a single mitochondria, this report is very important to novel type of eukaryotic gene expression. Successful yeast cell transformation in this report implies possible eukaryotic mitochodrial transformation including plants and animals and moreover two or more gene expression which can be excellent applicable protocols to pharmaceutical field including antibody production.

• The Korean Journal of Mycology
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• v.17 no.1
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• pp.27-30
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• 1989

F. velutipes Leu 2 gene (${\beta}-isopropylmalate$ dehydrogenase gene) was used for transformation of P. florida leucine requiring auxotrophic mutant P101. Transformation frequency was very low but the transformed colony can grow on minimal medium very slowly. Transformation was identified by Southern hybridization and reverse transformation into E. coli using chromosome DNA isolated from transformed P. florida.

• Animal cells and systems
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• v.2 no.3
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• pp.377-377
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• 1998