Inactivation of Agrobacterium tumefaciens Inoculated on Fresh Radix Ginseng by Electron Beam Irradiation and Aqueous Chlorine Dioxide Treatment

( Ho Hyun Chun ),( Ju Yeon Kim ),( Kyung Bin Song ) 한국응용생명화학회 2007 Journal of Applied Biological Chemistry (J. Appl. Vol.50 No.3

Inactivation of Agrobacterium tumefaciens was evaluated on the inoculated fresh Radix Ginseng by electron beam irradiation or aqueous chlorine dioxide (ClO2) treatment. Two groups of fresh ginsengs were prepared and inoculated with A. tumefaciens. One group was then irradiated at 0, 2, and 4 kGy using an electron beam accelerator, and the other group was treated with 0, 50, and 100 ppm of aqueous ClO2. Microbiological data indicated that populations of A. tumefaciens significantly decreased with increasing irradiation dose or aqueous ClO2 concentration. In particular, A. tumefaciens was eliminated by irradiation at 4 kGy, and 100 ppm ClO2 treatment reduced the populations of A. tumefaciens by 1.44 log CFU/g. These results suggest that electron beam irradiation or aqueous ClO2 treatment can be useful in improving the microbial safety of fresh ginsengs during storage.

Identification of Lettuce Genotypes to be Readily Transformed by Agrobacterium

KIM, ZHOO HYEON,KANG, SEONG MO,PYO, JAE SOO 경상대학교 유전공학연구소 1990 遺傳工學硏究所報 Vol.9 No.-

Highly-susceptible lettuce genotypes to Agrobacterium tumefaciens were screened. Leaf, stem and flower buds were easily infected by Agrobacterium. Upon inoculation, the upper parts of a stem responded better by exhibiting a higher tumorigenecity with bigger tumors. All accession lines inoculated were infected by Agrobacterium tumefaciens and formed tumors. However, percent tumorigenesis of the accessions ranged from 30 to 100, and mean tumor dry weight ranged from 0.01 to 1.58 gr. Almost all lines responded better by an Agrobacterium strain 15955 than by C58 in tumor size and percent tumorigenesis. Ten out of 62 lines responded especially well to Agrobacterium tumefaciens. The highly susceptible lines, 18, 52, and 63, could in a food crop to apply gentic engineering techniques for improvement.

재조합 Agrobacterium tumefaciens NTL4 균주를 이용한 quorum Sensing Autoinducer 검색에 용매와 계면활성제가 미치는 영향

고경표,김연희,김정선,박성훈,Koh, Kyong-Pyo,Kim, Yeon-Hee,Kim, Jung Sun,Park, Sunghoon 한국해양바이오학회 2006 한국해양바이오학회지 Vol.1 No.4

재조합 Agrobacterium tumefaciens균주를 사용한 Liquid Culture Assay는 quorum sensing autoinducer를 검색하는 생물학적 분석 방법으로 개발되었다. 그러나 이 시스템에 사용되는 해양천연물 시료들이 일반적으로 낮은 수용액에 대한 용해도를 갖기 때문에 활성검색에 걸림돌이 되고 있다. 시료의 용해도는 유기용매 혹은 계면활성제의 첨가로 증가될 수 있으나, 유기용매나 계면활성제 자체가 검색결과의 정확한 해석을 방해할 수 있으므로 적절한 조건의 확립이 매우 중요하다. Methanol, ethanol, 1-propanol, DMSO와 DMF를 0~10% 농도 범위에서 재조합 A. tumefaciens균주에 대한 세포 성장에 미치는 영향과 ${\beta}$-galactosidase activity에 미치는 영향을 살펴본 결과 methanol 2%이하의 농도가 가장 적합하였으며, 계면활성제의 경우, Tween 20, Tween 80보다는 Triton X-100이 약 0.05% 농도에서 세포내로의 활성물질의 전달효율을 높이는데 효과적이었다. The Liquid Culture Assay using a recombinant Agrobacterium tumefaciens strain has been developed as a means for quorum sensing autoinducer screening. However, the low aqueous solubility of marine natural product extracts used as potential autoinducers has been a hindrance in the screening process. Although the addition of organic solvents and/or surfactants could increase aqueous solubility, errors in data interpretation including false positive results could be a serious problem. Therefore, determining the best possible solvent and surfactant at the optimum concentration is crucial. Evaluating methanol, ethanol, 1-propanol, DMSO and DMF at concentration ranges of 0~10% revealed < 2% methanol to be most favorable when tested for ${\beta}$-gal activity and growth inhibition of the recombinant A. tumefaciens strain. On the other hand, among surfactants tested, Triton X-100 was similarly effective in increasing the delivery of autoinducers for activity at less than 0.05% concentration.

Efficiency of transformation mediated by Agrobacterium tumefaciens using vacuum infiltration in rice (Oryza sativa L.)

Safitri, Fika Ayu,Ubaidillah, Mohammad,Kim, Kyung-Min The Korean Society of Plant Biotechnology 2016 식물생명공학회지 Vol.43 No.1

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.

Insertion Mutation in HMG-CoA Lyase Increases the Production Yield of MPA through Agrobacterium tumefaciens-Mediated Transformation

( Yuguo Dong ),( Jian Zhang ),( Rui Xu ),( Xinxin Lv ),( Lihua Wang ),( Aiyou Sun ),( Dongzhi Wei ) 한국미생물 · 생명공학회 2016 Journal of microbiology and biotechnology Vol.26 No.11

Mycophenolic acid (MPA) is an antibiotic produced by Penicillium brevicompactum. MPA has antifungal, antineoplastic, and immunosuppressive functions, among others. β-Hydroxy-β-methylglutaryl-CoA (HMG-CoA) lyase is a key enzyme in the bypass metabolic pathway. The inhibitory activity of HMG-CoA lyase increases the MPA biosynthetic flux by reducing the generation of by-products. In this study, we cloned the P. brevicompactum HMG-CoA lyase gene using the thermal asymmetric interlaced polymerase chain reaction and gene walking technology. Agrobacterium tumefaciens-mediated transformation (ATMT) was used to insert a mutated HMG-CoA lyase gene into P. brevicompactum. Successful insertion of the HMG-CoA lyase gene was confirmed by hygromycin screening, PCR, Southern blot analysis, and enzyme content assay. The maximum MPA production by transformants was 2.94 g/l. This was 71% higher than wild-type ATCC 16024. Our results demonstrate that ATMT may be an alternative practical genetic tool for directional transformation of P. brevicompactum.

Agrobacterium으로 형질전환시킨 갈퀴꼭두선이의 세포배양에 의한 천연염료생산

신순희,김유선,김승혜 德成女子大學校 藥學硏究所 1993 藥學論文誌 Vol.4 No.1

The cells of Rubia cordifolia var. pratensis were transformed by Agrobactrium tumefaciens strain 11157. Surface-sterilized young leaves and stems of the plants were cocultivated with bacterial suspensions. Crown galls induced from stems were cultured with variation of culturing conditions and compared with untransformed cells. The growth rates and production of anthraquinone pigments of cells were remarkably improved by transformation. Furthermore, hairy roots were induced by inoculation or cocultivation with Agrobacterium rhizoigenes strains.

Agrobacterium 매개에 의한 고구마 형질전환 및 식물체 재분화

임순,양경실,권석윤,백기엽,곽상수,이행순,Lim, Soon,Yang, Kyoung-Sil,Kwon, Suk-Yoon,Paek, Kee-Yoeup,Kwak, Sang-Soo,Lee, Haeng-Soon 한국식물생명공학회 2004 식물생명공학회지 Vol.31 No.4

Transformed sweetpotato (Ipomoea batatas (L.) Lam. cv. Yulmi) plants were developed from embryogenic calli following Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105/pCAMBIA2301 harboring genes for intron $\beta$-glucuronidase (GUS) and kanamycin resistance. Transient expression of GUS gene was found to be higher when embryogenic calli were co-cultivated with Agrobacterium for 2 days. The co-cultured embryogenic calli transferred to selective MS medium containing 1mg/L 2,4-D, 100mg/L kanamycin, and 400mg/L claforan. These embryogenic calli were subcultured to the same selection medium at 4 weeks interval. Kanamycin-resistant calli transferred to hormone-free MS medium with kanamycin gave rise to somatic embryos and then converted into plantlets in the same medium. Southern blot analysis confirmed that the GUS gene was inserted into the genome of the sweetpotato plants. A histochemical assay revealed that the GUS gene was preferentially expressed in the leaf, petiole, and vascular tissue and tip of root. 국내 고구마 율미 품종의 배발생 캘러스를 Agrobacterium 매개 방법을 이용하여 형질전환 식물체를 개발하였다. 배발생 캘러스를 7일 동안 전배양 한 후 Agrobacterium과 2일 간 공동배양할 경우 일시적인 형질전환 효율이 가장 높았다. Agrobacterium과의 공동배양 후 배발생 캘러스를 1mg/L 2,4-D, 100mg/L kanamycin, 400mg/L claforan 이 첨가된 선발배지에서 4주 간격으로 계대배양하였다. 선발된 kanamycin 저항성 캘러스를 2,4-D를 제거한 선발배지로 옮겨 체세포배를 유도하였으며 이후 소식물체로 발달하였다. Southern 분석으로 1-3 copy의 GUS 유전자가 고구마 염색체내로 도입되었음을 확인하였다. 또한 조직학적 분석으로 GUS 유전자가 형질전환 고구마의 배발생 캘러스, 재분화 식물체의 잎, 엽병, 및 뿌리 조직에서 강하게 발현됨을 알 수 있었다.

Development of Auxotrophic Agrobacterium tumefaciens AGL1 by Tn5 Transposon for Rice (Oryza sativa L.) Transformation

Mohamed Sahrul Tamzil,Yuzer Alfiko,Andhika Faisal Mubarok,Sigit Purwantomo,Suwanto Antonius,Budiarti Sri 한국생물공학회 2021 Biotechnology and Bioprocess Engineering Vol.26 No.4

Explant contamination due to Agrobacterium overgrowth after the co-cultivation stage is a common problem in Agrobacterium-mediated plant transformation. In order to overcome this issue, this research undertook another approach by generating auxotrophic Agrobacterium tumefaciens AGL1 mutants to a specific amino acid by mini Tn5 transposon carrying spectinomycin resistance gene (spcR), and a total of 3315 AGL1 mutants were successfully constructed. Further screening identified 20 putative auxotrophs, and subsequently produced three mutants carried auxotroph properties to one specific amino acid. These mutants were AP5-2-51 threonine auxotroph, AP5- 5-2 cysteine auxotroph, and AP5-7-27 tryptophan auxotroph. The mini Tn5 insertion position in the Agrobacterium genome showed that the insertion position of AP5-2-51 mutants was in the thrB gene (AAK86584.1; locus tag At1D132_04580), while the other two mutants were unable to be identified by TAIL-PCR technique. The effectiveness of these three mutants to transfer T-DNA (pCAMBIA1300- eGFP-hpt) was examined on fresh Nipponbare rice callus explants with AGL1 as control. Results showed that transformation efficiency of the three mutants was not significantly different from AGL1 (Tukey HSD, α = 0.05). The percentages of Agrobacterium overgrowth in control and samples (three mutants) were also measured. Interestingly, the AP5-2-51 mutant indicated the highest ability to prevent overgrowth by reducing Agrobacterium growth to 1.11%, while the other two mutants suppressed the overgrowth to 15.56% (AP5-5-2) and 12.22% (AP5-7-27).

Agrobacterium tumefaciens-Mediated Genetic Transformation: Mechanism and Factors

Nitish Kumar,K.G. Vijayanand,Muppala P. Reddy,Amritpal S. Singh,Subhash Naraynan 강원대학교 산림과학연구소 2009 Journal of Forest Science Vol.25 No.3

Agrobacterium–mediated genetic transformation has been widely used for the production of genetically modified transgenic plants to obtain specific desired traits. Most of the molecular mechanisms that underlie the transformation steps have been well elucidated over the years. However, a few steps, such as nuclear targeting, T-DNA integration, and Agrobacterium-plant proteins involved remain largely obscure and are still under extensive studies. This review describes the major steps involved in the molecular mechanism of Agrobacterium-mediated transformation and provides insight in the recent developments in studies on the Agrobacterium-mediated genetic transformation system. Some factors affecting the transformation efficiency are also briefly discussed.

Agrobacterium tumefaciens-Mediated Genetic Transformation: Mechanism and Factors

Kumar, Nitish,Vijayanand, K.G.,Reddy, Myppala P.,Singh, Amritpal S.,Naraynan, Subhash Institute of Forest Science 2009 Journal of Forest Science Vol.25 No.3

Agrobacterium-mediated genetic transformation has been widely used for the production of genetically modified transgenic plants to obtain specific desired traits. Most of the molecular mechanisms that underlie the transformation steps have been well elucidated over the years. However, a few steps, such as nuclear targeting, T-DNA integration, and Agrobacterium-plant proteins involved remain largely obscure and are still under extensive studies. This review describes the major steps involved in the molecular mechanism of Agrobacterium-mediated transformation and provides insight in the recent developments in studies on the Agrobacterium-mediated genetic transformation system. Some factors affecting the transformation efficiency are also briefly discussed.