• Biotechnology and Bioprocess Engineering:BBE
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• 제9권3호
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• pp.217-222
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• 2004

As Agrobacterium tumefaciens, which has long been used to transform plants, is known to transfer T-DNA to budding yeast, Saccharomyces cerevisiae, a variety of fungi were subjected to the A. tumefaciens-mediated transformation to improve their transformation frequency and feasibility. The A. tumefaciens-mediated transformation of chestnut blight fungus, Cryphonectria parasitica, is performed in this study as the first example of transformation of a hardwood fungal pathogen. The transfer of the binary vector pBIN9-Hg, containing the bacterial hygromycin B phosphotransferase gene under the control of the Aspergillus nidulans trpC promoter and terminator, as a selectable marker, led to the selection of more than 1,000 stable, hygromycin B-resistant transformants per 1${\times}$10$\^$6/ conidia of C. parasitica. The putative transformants appeared to be mitotically stable. The transformation efficiency appears to depend on the bacterial strain, age of the bacteria cell culture and ratio of fungal spores to bacterial cells. PCR and Southern blot analysis indicated that the marker gene was inserted at different chromosomal sites. Moreover, three transformants out of ten showed more than two hybridizing bands, suggesting more than two copies of the inserted marker gene are not uncommon.

• Journal of Plant Biotechnology
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• 제4권4호
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• pp.135-141
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• 2002

Agrobacterium-mediated transformation has been unsuccessful for monocot plants except for a few important crops such as barley, rice, maize and wheat. We discussed here that a successful transformation of monocots demands certain critical conditions. The requirements for an efficient transformation are a selection of target tissues competent for plant regeneration and Agrobacterium-infection, and various factors promoting Agrobacterium-infection. The factors were divided into two to activate Agrobacterium and to increase plant cell's susceptibility against Agrobacterium. Optimization of these factors significantly increased transformation efficiency of zoysia grass and rice plants. A technical improvement in transformation system for monocots will promote improvement of the breed as well as a study of gene functions in monocots.

• Journal of Plant Biotechnology
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• 제5권1호
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• pp.19-23
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• 2003

The Ac (activator) which is one of the well-characterized transposable elements from maize was examined for its transposition possibility to the heterologous plant (P.nigra x maximowiczii) genome via Agrobacterium tumefacience (LBA4404) mediated transformation system. A number of transgenic plants were successfully recovered after 30 weeks by amount reduction from 50 to 15 g/$m\ell$ kanamycin for in vitro selection to minimize phytotoxic effects and to increase callus growth and regeneration efficiency. Among transgenic plants, 62 out of 106 transgenic poplars (58.5%) showed abnormal phenotypes such as severe serrated leaves and light leaf coloration. Indigo staining with X-gluc proved indirectly the restoration of Gus enzyme function and the presence of Ac in poplar genome by PCR. Southern analysis indicated the transposition and existence of Ac element in poplar genomes. In this research, an Agrobacterium-mediated transformation system in poplar species was developed and identified that Ac derived from maize can be excised and trans posed into other poplar genomes.

• Journal of Plant Biotechnology
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• 제49권1호
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• pp.61-73
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• 2022

In this study, we developed a reliable and efficient Agrobacterium-mediated genetic transformation system by applying sonication and vacuum infiltration to six chickpea cultivars (ICCV2, ICCV10, ICCV92944, ICCV37, JAKI9218, and JG11) using embryo axis explants. Wounded explants were precultured for 3 days in shoot induction medium (SIM) before sonication and vacuum infiltration with an Agrobacterium suspension and co-cultivated for 3 days in co-cultivation medium containing 100 µM/l of acetosyringone and 200 mg/l of L-cysteine. Responsive explants with putatively transformed shoots were selected using a gradual increase in kanamycin from 25 mg/l to 100 mg/l in selection medium to eliminate escapes. Results showed optimal transformation efficiency at a bacterial density of 1.0, an optical density at 600 nm wavelength (OD₆₀₀), and an infection duration of 30 min. The presence and stable integration of the β-glucuronidase (gusA) gene into the chickpea genome were confirmed using GUS histochemical assay and polymerase chain reaction. A high transformation efficiency was achieved among the different factors tested using embryo axis explants of cv. JAKI 9218. Of the six chickpea cultivars tested, JAKI9218 showed the highest transformation efficiency of 8.6%, followed by JG11 (7.2%), ICCV92944 (6.8%), ICCV37 (5.4%), ICCV2 (4.8%), and ICCV10 (4.6%). These findings showed that the Agrobacterium-mediated genetic transformation system will help transfer novel candidate genes into chickpea.

• 아시안잔디학회지
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• 제18권3호
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• pp.141-148
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• 2004

다양한 Zoysiagrass 4가지 품종들을 식물재료로 사용하여 Agrobacterium만 이용한 방법 그리고 particle bombardment로 배발생캘러스에 상처를 낸 후, Agrobacterium으로 공동배양 시키는 2가지 다른 형질전환 방법을 비교하였다. 예비실험에서 일반적으로 형질전환에 널리 사용되는 kanamycin과 PPT(phospinitricin)의 적적선발농도에 대해서 실험하였는데, kanamycin의 경우 300mg/l 그리고 PPT의 경위 50mg/l의 농도에서 가장 효과적인 선발 효율을 나타내었다. Agrobacterium을 이용한 형질전환은 Agrobacterium을 2일간 배양시킨 다음, 박테리아 농도를 O.D 600nm=1.0-1.2로 맞추고, 배발생캘러스를 30분간 간염 시키는 방법이 효과적이었는데, particle bombardment를 이용하여 캘러스에 상처를 유발시킨 후, Agrobacterium으로 감염시키면 3배 이상 높은 형질전환 수율을 획득할 수 있었다. 이상의 결과는 한국잔디 형질전환에 있어서 particle bombardment과 Agrobacterium을 병행하여 실시한 최초의 보고이고, 이러한 시스템을 기반으로 하여 향후 한국잔디를 포함하여 다른 난지형 및 한지형 잔디의 품종개량에 널리 이용되리라 생각된다.

• 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.

• 생명과학회지
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• 제11권4호
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• pp.316-320
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• 2001

Sonication tremendously improves the efficiency of Agrobacterium infection by introducing small and uniform fissures and channels throughout the targeted tissue. Using shoot tips of cotton as explants, the effect of sonication treatment and virulence genes in Agrobacterium tumefaciens on transformation efficiency was investigated. The pat gene which encodes resistance to the herbicide, glufosinate, was used as a selectable marker. Transformation efficiency was evaluated on th basis of survival rates of cocultivated shoot tips on selection medium containing 2.5 mg/l gulfosinate-ammonium(ppt) adn 25. mg/l Clavamax. Sonication from 5 to 15 second has a positive effect on shoop tip survival. However, whil virE as well as virG or vir GN54D showed an enhancement in transformation efficiency, virE,. virG resulted in the most significant enhancement. Overall, the combination of additional virG/virE gene and sonication treatment resulted in the most significant increase in transformation efficiency.

• Journal of Plant Biotechnology
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• 제32권4호
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• pp.275-279
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• 2005

Brassica campestris var. narinosa는 새싹채소로 애용되는 식물로서 간단한 용기에서 손쉽게 재배할 수 있다. 본 연구에서는 vacuum-infiltration을 통한 Agrobacterium-mediated transformation에 의해서 B. campestris var. narinosa 싹에서의 GUS일시발현을 성공적으로 확인하였다. 발아전의 종자가 발아를 이미 시작한 종자보다 그 형질전환 효율이 높게 나타났다. 한편, hydrogen peroxide를 2일 생장시킨 싹에 처리한 후 형질전환 하였을 때 GUS발현이 증가되는 것을 관찰하였다. 간염항원유전자의 형질전환과 ELISA에 의한 항원단백질의 발현량 분석 시 hydrogen peroxide 처리 싹이 비처리 형질전환 싹에서 보다 2배 이상의 발현량이 측정되었다.

• 원예과학기술지
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• 제18권6호
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• pp.797-801
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• 2000

본 연구는 고추에서 Agrobacterium tumefaciens를 이용한 형질 전환시 형질전환율을 높이기 위하여 A. tumefaciens와의 접종시간과 인위적인 상처처리에 따른 효과를 조사하였던 바 고추 형질전환시 자엽절편체의 재분화는 인위적 상처처리 및 A. tumefaciens 접종시간에 의해 영향을 받았다. 접종시간을 3600초로 한 경우의 재분화는 상처처리에 의해 급격히 감소하였으며 자엽 절편체에 대한 인위적인 상처처리가 20초 또는 120초간의 접종시간처리와 병행됨으로써 형질 전환율을 58-72% 이상으로 높이는 효과를 보였다. 형질전환여부의 확인을 ${\beta}$-glucuronidase 염색법과 npt II primer를 이용한 polymerase chain reaction 분석에 의해 수행 한 바 효과적인 것으로 판단되었다.

• Applied Biological Chemistry
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• 제51권4호
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• pp.342-343
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• 2008