• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2018년도 춘계학술발표회
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• pp.36-36
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• 2018

Soybean is the important crop in Asian countries as protein source, oil production and animal feed. Improving soybean using genetic transformation is the principal tool in nowadays. Developing herbicide resistant transgenic soybean plants through Agrobacterium-mediated transformation has been worked in many previous studied. However, the transformation efficiency is still low. Many attempts try to find the optimum media condition for plant regeneration after infection. After transformation, the plant regeneration is very important condition to promote growth of transgenic plant. In this study, we optimized a regeneration condition for two Korean soybean cultivar, Dawonkong and Pungsannamulkong using cotyledon, cotyledonary nodes and hypocotyl as explant. The results showed that shoot regeneration of cotyledonary nodes on B5 medium containing 2 mg/L 6-benzylaminopurine showed the highest percentage of regeneration in Dawonkong (75.8%) while Pungsannamulkong presented high number of shoots 2.12 shoots per explant. For transformation condition, co-cultivation in 7 days showed a high number of GUS positive expression. Most of explants can survived under media including 5 mg/L of glufocinate which refers phosphinotricin for 2-week selection. Washing with 400 mg/L of cefotaxime in several times and selection in plant regeneration media with 400 mg/L of cefotaxime can prevent bacteria growth, effectively.

• Journal of Crop Science and Biotechnology
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• 제10권2호
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• pp.92-97
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• 2007

Antibiotics used for suppressing Agrobacterium in plant transformation procedure might have negligible effects on plant tissues and regeneration. The effects of antibiotics on growth suppression of Agrobacterium and plant regeneration were investigated for enhancing Agrobacterium-mediated transformation using wheat mature embryos. Antibiotics tested, except carbenicillin, were able to suppress that embryos were coated with a layer of Agrobacterium cells in callus induction medium. Agrobacterium growth was suppressed minimally at 50 mg/l of timentin, while cefotaxime and clavamox were completely suppressed at relative high concentration of 250 mg/l. In the treatment of carbenicillin, initiation of growth suppression of Agrobacterium occurred at 750 mg/l of concentration because Agrobacterium KYRT1 contains the carbenicillin resistant gene. In Agrobacterium inoculation, effects of antibiotics were significantly different on the rate of callus induction and shoot formation. Almost embryos were induced calli at 50 mg/l of timentin whereas callus induction rate was achieved above 90% at 100 mg/l and 250 mg/l of cefotaxime and clavamox, respectively. Shoot formation rate was higher in the treatment of timentin than that of cefotaxime and clavamox at 500 mg/l of concentration, respectively. Timentin can be used as a good antibiotics in Agrobacterium-mediated wheat transformation.

• Plant Resources
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• 제7권1호
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• pp.39-46
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• 2004

To demonstrate the importance of transformation efficiency in independent event, molecular and cytogenetic analysis were conducted with genomic DNA and chromosome of transgenic plants produced by Agrobacterium tumefeciens LBA4404 (pSBM-PPGN: gusA and bar). Selection ratios of putative transgenic calli were similar in independent experiments, however, transformation efficiencies were critically influenced by the type of regeneration media. MSRK5SS-Pr regeneration mediun, which contains 5 mgL$^{-1}$ kinetin, 2% (w/v) sucrose in combination with 3% (w/v) sorbitol, and 500 mgL$^{-1}$ proline, was efficient to produce transgenic plant of rice from putative transgenic callus in the presence of L-phosphinotricin (PPT). With MSRK5SS-Pr medium, transformation efficincies of Nagdongbyeo were significantly enhanced from 3.7% to 6.3% in independent callus lines arid from 7.3% to 19.7% in plants produced, respectively. Stable integration and expression of bar gene were confirmed by basta herbicide assay, PCR amplification and Southern blotting of bar gene, and fluorescence in situ hybridization (FISH) analysis using pSBM-PPGN as a probe. In Southern blot analysis, diverse band patterns were observed in total 44 transgenic plants regenerated from 20 independent PPT resistant calli showing from one to five copies of T-DNA segments, however, the transformants obtained from one callus line showed the same copy numbers with the same fractionized band patterns.

• Mycobiology
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• 제45권2호
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• pp.84-89
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• 2017

Fungi of the Metarhizium genus are a very versatile model for understanding pathogenicity in insects and their symbiotic relationship with plants. To establish a co-transformation system for the transformation of multiple M. robertsii genes using Agrobacterium tumefaciens, we evaluated whether the antibiotic nourseothricin has the same marker selection efficiency as phosphinothricin using separate vectors. Subsequently, in the two vectors containing the nourseothricin and phosphinothricin resistance cassettes were inserted eGFP and mCherry expression cassettes, respectively. These new vectors were then introduced independently into A. tumefaciens and used to transform M. robertsii either in independent events or in one single co-transformation event using an equimolar mixture of A. tumefaciens cultures. The number of transformants obtained by co-transformation was similar to that obtained by the individual transformation events. This method provides an additional strategy for the simultaneous insertion of multiple genes into M. robertsii.

• 한국작물학회지
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• 제49권5호
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• pp.415-418
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• 2004

The effects of sonication and vacuum infiltration on transformation efficiency was investigated by using immature embryos of Korean wheat as explants. Two Agrobacterium tumefaciens strains, KYRT1 and EHA105, carrying pCAMBIA 1305.1 were used. Transformation efficiency was demonstrated by the detection of $\beta-glucu-ronidase$ (GUS) activity. GUS expression showed clear difference among Korean wheat cultivars. Geurumil showed higher GUS expression efficiency $79.1\%$ compared with other cultivars. The effects of the duration of vacuum infiltration and sonication treatment showed a tendency high GUS expression efficiency by their combination. In comparison with other Agrobacterium strains, KYRT1 showed high efficiency in most Korean cultivars.

• Journal of Plant Biotechnology
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• 제31권1호
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• pp.13-17
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• 2004

형질전환 효율을 높이기 위하여, NPTII와 GUS유전자를 포함하고 있는 pIG121Hm을 미세 입자에 코팅하여 particle bombardment를 수행한 후 pIG121Hm가 도입된 Agrobacterium tumefaciens EHA101와 3일간 공동 배양하였다. 그 후 캘러스를 1mg/L 2,4-D, 0.1mg/L BAP, 100mg/L kanamycin, 그리고 200mg/L carbenicillin이 포함된 MS배지로 옮겨 4주간 성장시킨 후 kanamycin저항성 캘러스를 선발하여 GUS 발현을 관찰하였고, PCR 분석을 통하여 700 bp의 NPT II 유전자가 도입된 것을 확인하였다. Particle bombardment 후 Agrobacterium과 공동배양 한 처리구가 Agrobacterium과 공동배양만 한 처리구보다 GUS발현 분석에 의한 형질전환 효율이 3배정도 더 높았다 형질전환 된 재분화 식물체를 얻기위해 다시 선발된 kanamycin저항성 캘러스는 1mg/L NAA와 1mg/L BAP가 포함된 재분화 배지에 옮겨져 4주 후 형질전환 된 소인경을 형성하였다.

• Mycobiology
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• 제43권1호
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• pp.1-8
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• 2015

Mushroom transformation requires a series of experimental steps, including generation of host strains with a desirable selective marker, design of vector DNA, removal of host cell wall, introduction of foreign DNA across the cell membrane, and integration into host genomic DNA or maintenance of an autonomous vector DNA inside the host cell. This review introduces limitations and obstacles related to transformation technologies along with possible solutions. Current methods for cell wall removal and cell membrane permeabilization are summarized together with details of two popular technologies, Agrobacterium tumefaciens-mediated transformation and restriction enzyme-mediated integration.

• Journal of Plant Biotechnology
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• 제6권2호
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• pp.69-75
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• 2004

Optimal protocol for efficient genetic transformation has been defined to aid future strategies of genetic engineering in pigeon pea with agronomically important genes. Transgenic pigeonpea plants were successfully produced through Agrobacterium tumefaciens-mediated genetic transformation method using cotyledonary node explants by employing defined culture media. The explants were co-cultivated with A. tumefaciens strain C-58 harboring the binary plasmid, pCAMBIA-1301 [con-ferring $\beta$-glucuronidase(GUS) activity and resistance to hygromycin] and cultured on selection medium (regeneration medium supplemented with hygromycin) to select putatively transformed shoots. The shoots were then rooted on root induction medium and transferred to pots containing sand and soil mixture in the ratio of 1:1. About 22 putative TO transgenic plants have been produced. Stable expression and integration of the transgenes in the putative transgenics were confirmed by GUS assay, PCR and Southern blot hybridization with a transformation efficiency of over 45%. Stable integration and expression of the marker gene has been confirmed in the TO and T1 transgenics through PCR, and Southern hybridization.

• Journal of Microbiology and Biotechnology
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• 제18권4호
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• pp.754-758
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• 2008

Agrobacterium tumefaciens-mediated transformation (ATMT) was successfully applied to Monascus ruber. The optimum cocultivation time was 84 h with an efficiency of 900 to 1,000 transformants when $1{\times}10^6$ spores were used with the same volume of bacteria. The stability of transform ants was over 98% after five generations. When M. ruber was transformed with A. tumefaciens YL-63 containing the green fluorescent protein gene (egfp), the green fluorescent signal was observed throughout hyphae, confirming expression of the gene. This efficient transformation and expression system of M. ruber by ATMT will facilitate the study of this fungus at a molecular genetic level.

• Journal of Plant Biotechnology
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• 제4권4호
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• pp.151-154
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• 2002

Conditions for lily pollen growth in vitro and transformation were optimized. Active pollen tube development was achieved effectively in a medium containing 7% sucrose with pH adjusted to 5.7 at the temperature of 27$^{\circ}C$ for about 16-24 hours. Pollen growth was little impaired by the presence of kanamycin at concentration up to 100 mg/L. Pollen rains near the beginning of germination stage were more reliable for Agrobacterium-mediated GUS DNA transformation via vacuum infiltration lasted for 20-40 minutes. GUS DNA integration and its expression in fully developed pollen tubes could be confirmed by Southern blot hybridization, RT-PCR and histochemical staining.