• Plant Resources
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• v.8 no.2
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• pp.87-90
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• 2005

In tobacco, in vitro pollination has been successfully applied to overcome interspecific incompatibility. The use of this technique will make it possible to introduce DNA into pollen tubes just before fertilization. In this study, we showed improvement of the efficiency of in vitro self-pollination and introduction of foreign genes into pollen tubes by the method of polycation. A plasmid harbouring the GUS gene was introduced into pollen grains and pollen tubes, which had incubated on pollen germination medium(PGM), by polyornithine method. Transient expression of the GUS in pollen grains and pollen tubes that were treated with 0, 2, 5 and $10{\mu}g/m\ell$ DNA was observed. In results, combination of the techniques of polyornithine and in vitro pollination was efficient new technique for genetic transformation through fertilization processes.

• Journal of Korean Society of Forest Science
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• v.87 no.2
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• pp.164-172
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• 1998

This study was conducted to find the optimum transformation condition using Agrobacterium harboring promoterless GUS gene. The optimal medium for shoot induction from leaves of Populus nigra${\times}$P. maximowiczii was MS medium supplemented with $0.1mg/{\ell}$ NAA, $0.5mg/{\ell}$ BAP(94% regeneration frequency and 11.5 average number of shoot) According to the test using pBI121, the concentration of antibiotics for selection marker gene was $100mg/{\ell}$ kanamycin or $60mg/{\ell}$ geneticin in the SIM(shoot inducing medium) 3. Two weeks later, callus was induced in the SIM 3 and this callus grew up to 0.5-1cm shoots after 6 weeks in the new SIM 3. And the treatment with methylation inhibitor(5-azacytidine) led to a dramatic increase in foreign gene expression rate from 5.7% to 26.7%. The vector systems showed. different transformation efficiencies based on the fluorometric and histochemical GUS assay. In this study the vector systems used for transformation seemed to affect transformation frequency, in which pEHA101 yielded more transformants(35.9%) than LBA4404/pBI121 did(5.7%). This result indicated that pEHA101 was effective to insert the promoterless foreign gene into a poplar genome.

• Journal of Practical Agriculture & Fisheries Research
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• v.25 no.4
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• pp.138-147
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• 2024

Agrobacterium-mediated transformation (AMT) is a method that allows for the stable integration of DNA fragments into the plant genome. Transgenic plants generated through AMT typically exhibit a lower copy number of the transgene compared to those induced by particle bombardment. Furthermore, AMT offers a straightforward and efficient approach for generating transgenic plants. While the transformation efficiency of wheat is comparatively lower than that of other monocot plants such as Rice (Oryza sativa L.) and Maize (Zea mays L.), the cultivars 'Bobwhites' and 'Fielder' are commonly employed for wheat transformation. To date, there have been no reported instances of successful development of transgenic plants using Korean wheat varieties through AMT. This study aims to assess the transformation efficiency of 43 Korean wheat cultivars using the GUS assay, with the goal of identifying suitable Korean wheat cultivars for AMT. The pCAMBIA1301 vector, carrying the β-glucuronidase (GUS) gene, was incorporated into Agrobacterium strain EH105. Following the inoculation of Agrobacterium into immature embryos, GUS assays were conducted 'Saeol', 'Jopum', and 'Jonong' showed 100% (the number of embryos showing GUS spots/the number of embryos used for AMT) among 43 cultivars. In addition, cultivars with more than 70% were 'Saekeumgang', 'Jojung', 'Tapdong', 'Anbaek', 'Dabun', 'Sugang', 'Keumgang', 'Jeokjung', 'Seodun', 'Joeun', 'Dajung', and 'Baekjung'. It seems that the 15 cultivars above showed the possibility of using AMT. On the other hand, 'Yeonbaek', 'Goso', 'Baekgang', and 'Johan' showed less than 20% and GUS spots were not observed in 'Gru', 'Gobun', 'Milseong', and 'Shinmichal-1'. This study explores transient GUS expression in Korean wheat cultivars seven days after AMT. The observed initial high efficiency of transient transformation suggests the potential for subsequent stable transformation efficiency. Korean wheat cultivars demonstrating elevated transient transformation efficiency could serve as promising candidates for the development of stable transgenic wheat.

• Journal of Applied Biological Chemistry
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• v.54 no.1
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• pp.26-32
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• 2011

To improve genetic transformation of Brassica napus winter cultivar 'Youngsan', factors influencing shoot regeneration and transformation from cotyledonary petioles were investigated. Shoot induction was enhanced in the combination of 0.5 mg/L NAA and 2~4 mg/L kinetin. Silver nitrate was essential for successful shoot regeneration, ranging from 5 to 9 mg/L. The addition of $GA_3$ promoted plant regeneration. Among the tested Agrobacterium strains, co-cultivation times, and antibiotic selection regimes, choice of appropriate Agrobacterium strain was the most critical factor for efficient transformation of B. napus cv. 'Youngsan'. The EHA105 succinamopine strain was the most efficient and the maximum transformation efficiency was 26.8%. Transgenic shoots were selected on 10 mg/L phosphinothricin (PPT) containing media. The transgenic plants expressing bar and gus genes were resistant for commercial herbicide "Basta" and stained with X-Gluc. Southern blot hybridization indicated that the presence of one to three gus gene copies per genome and inheritance of the gus gene into the $T_1$ generation.

• Journal of Plant Biotechnology
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• v.33 no.4
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• pp.271-276
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• 2006

Hypocotyl explants of Chinese cabbage (us. 'Jeong Sang' and 'Seoul') produced adventitious shoots on Murashige and Skoog (MS) basal medium supplemented with 4mg/L $AgNO_3$, 5 mg/L acetosyringone, 4 mg/L 6-benzyladenine and 3mg/L alpha-naphthaleneacetic acid (SI) after cocoultivation with strains of Agrobacterium tumefaciens (LBA4404) harboring the pCAMBIA1301 and the $_PPTN290$ containing hygromycin-resistance gene and paromomycin-resistance gene as a selectable marker genes, respectively. There was a significant difference in the frequency of transgenic plants depending on antibiotics and cultivars used. Paromomycin was better than hygromycin, and cultivar 'Jeong-sang' was higher than 'c.v. Seoul' in the frequency of transgenic plants. In particular, the highest frequency (0.70%) of transgenic plants was obtained from selection medium (SI) containing 100mg/L paromomycin in c.v., 'Jeong-sang' GUS positive response were obtained 9 plants and 3 plants from the cultivars, 'Jeong-sang' and 'Seoul', respectively. They were grown to maturity in a greenhouse and normally produced $T_1$ seeds. GUS histochemical assay for progeny $(T_1)$ revealed that the transgenes were expressed in the plant genome.

• Molecules and Cells
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• v.27 no.4
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• pp.475-480
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• 2009

The transcription of soybean (Glycine max) calmodulin isoform-4 (GmCaM-4) is dramatically induced within 0.5 h of exposure to pathogen or NaCl. Core cis-acting elements that regulate the expression of the GmCaM-4 gene in response to pathogen and salt stress were previously identified, between -1,207 and -1,128 bp, and between -858 and -728 bp, in the GmCaM-4 promoter. Here, we characterized the properties of the DNA-binding complexes that form at the two core cis-acting elements of the GmCaM-4 promoter in pathogen-treated nuclear extracts. We generated GUS reporter constructs harboring various deletions of approximately 1.3-kb GmCaM-4 promoter, and analyzed GUS expression in tobacco plants transformed with these constructs. The GUS expression analysis suggested that the two previously identified core regions are involved in inducing GmCaM-4 expression in the heterologous system. Finally, a transient expression assay of Arabidopsis protoplasts showed that the GmCaM-4 promoter produced greater levels of GUS activity than did the CaMV35S promoter after pathogen or NaCl treatments, suggesting that the GmCaM-4 promoter may be useful in the production of conditional gene expression systems.

• Korean Journal Plant Pathology
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• v.11 no.1
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• pp.66-72
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• 1995

The coat protein (CP) gene of cucumber mosaic virus-As (CMV-As) strain was engineered for expression in the plant by using the cauliflower mosaic virus 35S transcript regulatory sequences. The CP gene was cloned into an Agrobacterium-derived binary vector. A chimeric gene was constructed by the cDNA of CMV-As CP and plant expression vector pBI121. The clone, pCMAS66, was first introduced into the phagemid vector pSPORT1 for situating sense orientation for translation and making restriction sites in order to re-introduce plant expression vector, pHI121. The resulting subclone pCASCP02 and plant expression vector pBI121 were treated with BamHI-SacI for excising the target gene and removing GUS gene, respectively. After Agrobacterium transformation by freeze-thaw technique, the clone, pCMASCP121-123 which contains sense orientation of the target gene, was selected and confirmed by restriction endonuclease analysis. The CMV-As CP gene was introduced into A. tumefaciens. The results on tobacco plant transformation with the vector system revealed that the system could be successfully introduced and showed high frequency of selection to putative transformations.

• Journal of Life Science
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• v.16 no.2 s.75
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• pp.289-296
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• 2006

In order to establish highly efficient gene transfer condition at early stage of soybean transformation, various experiments were performed and compared their efficiencies by transient GUS analysis; those conditions are genotype determination of Korean soybean cultivars for amenability to Agro-infection, appropriate agar and selective agent concentration, orientation of explant placement, hormone pre-culture, and liquid selection condition. In the genotype screen of Korean soybean varieties, 14 amenable genotypes were selected. For efficient Agrobacterium washing, cefotaxime was chosen and hygromycin at the concentration of 10 and 15 ppm was used as selection agent in the media. Agar concentration was slightly better in 0.6% and 0.8% for both shoot and callus formation, and explant placement with adaxial side down showed high frequency of GUS expression. For wounding treatment, oriental needle was efficient than scalpel for shoot formation and gene transfer. To increase the frequency of gene transfer, hormone pre-treatment was applied. BA at the concentration of 5 and 10 ppm resulted in better survival at the late stage of selection in shoot elongation media. Selection in liquid media after hormone pre-treatment seemed to be effective to remove the escaped non-transformants at early stage of procedure. Considering the results obtained, Eunhakong could be the first choice as a material for soybean transformation among Korean soybean genotypes.

• Korean Journal of Breeding Science
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• v.41 no.3
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• pp.252-260
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• 2009

Rice is the most important cereal crop not only in supplying the basic staple food for more than half of the world's population but also as a model plant for functional genomic studies of monocotyledons. Although rice transformation method using A. tumefaciens has already been widely used to generate transgenic plants, the transformation rate is still low in most Korean elite cultivars. We made several modifications of the standard protocol especially in the co-cultivation step to improve the efficiency of the rice transformation. The co-culture medium was modified by the addition of three antioxidant compounds (10.5 mg/L L-cysteine, 1 mM sodium thiosulfate, 1 mM dithiothreitol) and of Agrobacterium growth-inhibiting agent (5 mg/L silver nitrate). Co-cultivation temperature ($23.5^{\circ}C$ for 1 day, $26.5^{\circ}C$ for 6 days) and duration (7 days) were also changed. The plasmid of pMJC-GB-GUS carrying the GUS reporter gene and the bar gene as the selectable marker was used to evaluate the efficiency of the transformation. After co-cultivation, a high level of GUS gene expression was observed in calli treated with the modified method. It is likely that those newly added compounds helped to minimize the damage due to oxidative bursts during plant cell-Agrobacterium interaction and to prevent necrosis of rice cells. And the transformation rate under the modified method was also remarkably increased approximately 8-fold in Heungnambyeo and 2-fold in Ilmibyeo as compared to the corresponding standard method. Furthermore, we could produce the transgenic plants stably from Ilpumbyeo which is a high-quality rice but its transformation rate is extremely low. Transformation and the copy number of transgenes were confirmed by PCR, bar strip and Southern blot analysis. The improved method would attribute reducing the effort and the time required to produce a large number of transgenic rice 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)