• Journal of Microbiology and Biotechnology
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• 제11권2호
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• pp.222-228
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• 2001

Vibrio metschnikovii strain RH530 is a non-pathogenic, industrially-important alkaline protease producer which has been isolated from wastewater. In this paper, we report on the transformation of this strain by using the method of electroporation. A field strength of $7.5\;kVcm^{-1}$ and $25\;{\mu}F$, and using a 0.2-cm cuvette, appeared to be the optimal conditions for electroporation of the cells with the recombinant pSBCm plasmid carrying the vapK alkaline protease gene and the ColE1 replicon. Cells were subjected to osmotic shock in order to remove extracelluar DNase, and adding 200 mM of sucrose to electroporation buffer cells showed an increased transformation efficiency. Maximum efficiency of transformation was obtained at an early exponential growth phase. Using all of the conditions mentioned above, we routinely obtained a transformation efficiency of more than $10^4{({\mu}g\;plasmid\;DNA)}^{-1}$. The stability of the plasmid pSBCm in V. metschnikovii RH530 was 25% after 18h of growth (27 generations) in the medium without antibiotic selection. The insertion of the par locus to the pSBCm increased the stability of the plasmid up to 42% without selective pressure. The increase in plasmid stability was accompanied by the increase in the productivity of alkaline protease in the recombinant V. metschnikovii strain RH530. Determining optimal conditions for the transformation of the industrially-important, nonpathogenic Vibrio strain, and the improvement of plasmid stability by introducing the par locus into the high copy number plasmid vector, will allow the development of procedures involved in the genetic manipulation of this strain, particularly for its use in the production of industrial enzymes such as alkaline protease.

• Journal of Plant Biotechnology
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• 제48권4호
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• pp.246-254
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• 2021

Environmental stresses are estimated to have reduced global crop yields of wheat by 5.5%. However, traditional approaches for the transfer of resistance to these stresses in wheat plants have yielded limited results. In this regard, genetic transformation has undoubtedly opened up new avenues to overcome crop losses due to various abiotic stresses. Particle bombardment has been successfully employed for obtaining transgenic wheat. However, most of these procedures employ immature embryos, which are not available throughout the year. Therefore, the present investigation utilized mature seeds as the starting material and used the calli raised from three Moroccan durum wheat varieties as the target tissue for genetic transformation by the biolistic approach. The pANIC-5E plasmid containing the SINA gene for drought and salinity tolerance was used for genetic transformation. To enhance the regeneration capacity and transformation efficiency of the tested genotypes, the study compared the effect of copper supplementation in the induction medium (up to 5 μM) with the standard MS medium. The results show that the genotypes displayed different sensitivities to CuSO₄, indicating that the transformation efficiency was highly genotype-dependent. The integration of transgenes in the T₀ transformants was demonstrated by polymerase chain reaction (PCR) analysis of the obtained resistant plantlets with primers specific to the SINA gene. Among the three genotypes studied, 'Isly' showed the highest efficiency of 9.75%, followed by 'Amria' with 1.25% and 'Chaoui' with 1%.

• Journal of Microbiology and Biotechnology
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• 제11권1호
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• pp.61-64
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• 2001

Delta-integration vectors were constructed for the purpose of achieving homologous integration of the hirudin expression cassette into the chromosome of Saccharomyces cerevisiae. A double $\delta$ system truncated with the unnecessary bacterial genes, and consequently having a reduced insert size for integration, showed a four-fold increase in transformation efficiency at given DNA concentrations, and as a result, the constructed recombinant yeast strain had a 1.3-fold enhancement in hirudin expression level compared with a single $\delta$ system.

• Journal of Plant Biotechnology
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• 제7권2호
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• pp.113-121
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• 2005

The objective of this study was to identify the major parameters controlling DNA delivery by particle bombardment to immature embryos of Chinese spring wheat (Triticum aestivum L.). Efficiency of DNA (uidA gene) delivery was assessed by transient GUS (${\beta}$-glucuronidase) expression in bombarded tissues. Of the parameters analyzed, acceleration pressure, bombardment distance, chamber vacuum pressure, bombardment times, osmotic conditioning of culture had a remarkable influence on transient gene expression. A bombardment procedure suitable for Chinese spring wheat cultivars was developed which allowed high-efficiency DNA delivery combined with reduced damage to target tissues. The high efficiency made the system practical for wheat genetic transformation research and accelerating wheat breeding programs.

• Biotechnology and Bioprocess Engineering:BBE
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• 제3권1호
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• pp.48-49
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• 1998

A protocol for the transformation of Klebsiella oxytoca by electroporation was developed. Preparation of competent cells at early exponential phase was most critical to obtain high transformation efficiency. The highest efficiency of 1.6$\times$106 transformants per $\mu\textrm{g}$ DNA(pBR322) could be obtained by electroporation of K. oxytoca cells prepared at the OD600 of 0.2 with 1.25$\mu\textrm{g}$ DNA at the filed strength of 2.5kV, the parallel resistance of 200$\Omega$ and capacitance of 25$\mu$F.

• 한국항만경제학회지
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• 제26권2호
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• pp.82-98
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• 2010

본 연구에서는 첫째, 퍼지DEA모형을 해운항만분야에 이용한 국내외 기존연구들을 간략하게 검토하였으며, 둘째, Campos and Gonzalez(1989), 임성묵(2008)의 평균지수변환모형을 이론적으로 소개하였으며, 셋째, 국내 26개항만을 대상으로 2개의 투입요소(접안능력, 하역능력), 2개의 산출요소(화물처리량, 입출항척수)를 이용하여 평균지수변환모형에 의거하여 효율성을 분석하고 해석하였다. 실증분석결과를 요약해 보면 다음과 같다. 첫째, 일반 투입지향 CCR모형에서는 통영, 고현, 옥포, 속초항이 효율적이었으며, 여수항이 90% 후반의 효율성을 보였다. 둘째, 퍼지DEA 평균지수변환모형에서는 고현, 속초항이 가장 효율적이었으며, 옥포, 여수항은 람다값이 커질수록 효율성이 증가되었다. 또한 완도, 여수, 서귀포항은 람다값이 높아질 수록 효율성수치도 높아졌다. 셋째, 일반적인 투입지향 CCR 모형의 효율성 수치와 평균지수변환법에 의한 효율성수치의 평균순위는 거의 일치하였다. 본 논문이 갖는 정책적인 함의는 국내항만의 정책입안담당자들은 투입요소와 산출요소의 값을 정확히 알지 못하고 애매모호한 수준에서 알고 있을 때, 본 논문에서 사용한 퍼지 DEA 평균지수모형을 이용할 필요성이 있다는 점이다.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2018년도 추계학술대회
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• pp.52-52
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• 2018

Development of herbicide resistant transgenic legume plants through Agrobacterium-mediated transformation has been worked in many previous studied. Plant regeneration after infection is the important step to obtain successful transgenic plants. Many attempts try to find the optimum media condition for plant regeneration after infection. However, the transformation efficiency of legume plants is still low. In this study, regeneration of some Korean legume species including two soybean cultivars (Dawon and Pungsan) and pea have been done with organogenesis which is used various kind of explants such as cotyledonary-nodes in soybean and bud-containing tissue in pea. We developed the optimum media condition for plant regeneration regulators under Agrobacterium-mediated transformation using different kind and various concentration of plant growth. As the results, B5 medium containing 2 mg/L of 6-benzylaminopurine was selected in this study for the optimum plant regeneration media. The segments were inoculated with Agrobacterium suspension harbored an IG2 vector containing bar gene which confers resistance to phosphinotricin (PPT) in 3, 5 and 7 days. The transformation efficiency was achieved in Dawon 3.03 % and pea 1.46 % with co-cultivation period of 7 days which is showed a high number of GUS positive expression period.

• Journal of Plant Biotechnology
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• 제43권1호
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• pp.66-75
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• 2016

Agrobacterium-mediated gene transfer has recently been developed to improve rice transformation. In this study, 3 different transformation methods were tested including soaking, co-cultivation, and vacuum infiltration. Agrobacterium tumefaciens GV3101 harboring the binary vector pGreen:: LeGSNOR was used in this experiment. This study aimed to identify the most appropriate method for transferring LeGSNOR into rice. Vacuum infiltration of the embryonic calli for 5 min in Ilpum resulted in high transformation efficiency based on confirmation by PCR, RT-PCR, and qRT-PCR analyses. In conclusion, we described the development of an efficient transformation protocol for the stable integration of foreign genes into rice; furthermore, the study results confirmed that PCR is suitable for efficient detection of the integrated gene. The vacuum infiltration system is a potentially useful tool for future studies focusing on transferring important genes into rice seed calli, and may help reduce time and effort.

• 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.

• 응용통계연구
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• 제17권1호
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• pp.27-34
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• 2004

Horvitz-Thompson(H-T)추정량이 확률비례추정량에 비해 효율이 떨어지는 경우가 있다 이를 극복하기 위해서 2단계 변수변환을 한다. 1단계로는 Midzuno-Sen추출을 적용하기 위해서 보조변수를 변환하고, 이로부터 얻은 포함확률을 H-T추정량에 사용할 때 분산을 줄이기 위해서 2단계로 연구변수를 변환하였다. 이러한 변환을 통해 얻은 추정량과 기존의 PPS 추정량들을 비교하였다.