• 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.

• 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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• 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.

• 한국작물학회지
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• 제49권5호
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• pp.434-439
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• 2004

Medicago truncatula is a model plant for molecular genetic studies of legumes and plant-microbe interactions. To accelerate finding of genes that play roles in the early stages of nodulation and stress responses, a trans-genic plant was developed that contains a promoter­reporter fusion. The promoter of rip], a Rhizobium-induced peroxidase gene, was fused to the coding region of $\beta-glucuronidase (GUS)$ gene and inserted into a modified plant transformation vector, pSLJ525YN, in which the bar gene was preserved from the original plasmid but the neomycin phosphotransferase gene was replaced by a polylinker. Transformation of M. truncatula was carried out by vacuum infiltration of young seedlings with Agrobacterium. Despite low survival rates of infiltrated seedlings, three independent transformants were obtained from repeated experiments. Southern blot analyses revealed that 7 of 8 transgenic plants of the T 1 generation contained the bar gene whereas 6 $T_1$ plants contained the GUS gene. These results indicate that vacuum infiltration is an effective method for transformation of M. truncatula. The progeny seeds of the transgenic plants will be useful for mutagenesis and identification of genes that are placed upstream and may influence the expression of rip] in cellular signaling processes including nodulation.

• 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.

• Journal of Plant Biotechnology
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• 제48권3호
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• pp.156-164
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• 2021

Spirodela polyrhiza, from the Lemnaceae family, are small aquatic plants that offer an alternative plant-based system for the expression of recombinant proteins. However, no turion transformation protocol has been established in this species. In this study, we exploited a pB7YWG2 vector harboring the eYFP gene that encodes enhanced yellow fluorescent protein (eYFP), which has been extensively used as a reporter and marker to visualize recombinant protein localization in plants. We adopted Agrobacterium tumefaciens-mediated turion transformation via vacuum infiltration to deliver the eYFP gene to turions, special vegetative forms produced by duckweeds to endure harsh conditions. Transgenic turions regenerated several duckweed fronds that exhibited yellow fluorescent emissions under a fluorescence microscope. Western blotting verified the expression of the eYFP protein. To the best of our knowledge, this is the first report of an efficient protocol for generating transgenic S. polyrhiza expressing eYFP via Agrobacterium tumefaciens-mediated turion transformation. The ability of turions to withstand harsh conditions increases the portability and versatility of transgenic duckweeds, favoring their use in the further development of therapeutic compounds in plants.

• Plant Biotechnology Reports
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• 제5권3호
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• pp.217-224
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• 2011

Non-heading Chinese cabbage (Brassica rapa L. ssp. chinensis) is a popular vegetable in Asian countries. The diamondback moth (DBM), Plutella xylostella (L.), an insect with worldwide distribution, is a main pest of Brassicaceae crops and causes enormous crop losses. Transfer of the anti-insect gene into the plant genome by transgenic technology and subsequent breeding of insect-resistant varieties will be an effective approach to reducing the damage caused by this pest. We have produced transgenic non-heading Chinese cabbage plants expressing the potato proteinase inhibitor II gene (pinII) and tested the pest resistance of these transgenic plants. Non-heading Chinese cabbages grown for 45 days on which buds had formed were used as experimental materials for Agrobacterium-mediated vacuum infiltration transformation. Forty-one resistant plants were selected from 1166 g of seed harvested from the infiltrated plants based on the resistance of the young seedlings to the herbicide Basta. The transgenic traits were further confirmed by the Chlorophenol red test, PCR, and genomic Southern blotting. The results showed that the bar and pinII genes were co-integrated into the resistant plant genome. A bioassay of insect resistance in the second generation of individual lines of the transgenic plants showed that DBM larvae fed on transgenic leaves were severely stunted and had a higher mortality than those fed on the wild-type leaves.

• KSBB Journal
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• 제19권3호
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• pp.174-177
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• 2004

기내에서의 발아 및 형질전환 활성을 지닐 수 있는 백합 (Lilium longiflorum) 화분의 장기보존조건을 얻기 위하여 용매를 이용한 연구를 수행하였다. 화분의 용이한 수집을 위하여 용매를 사용했을 때, petroleum ether, n-heptane, benzene 등의 경우 화분의 기내발아에는 영향을 미치지는 않았으나 수집과 정 후 바로 화분건조 및 냉동 보관함으로써 발아활성을 유지 할 수 있었다. 이들의 활성유지는 또한 vacuum infiltration 및 Agrobacterium을 이용한 형질전환에 따른 외래유전자인 tissue plasminogen activator의 발현에 의하여 확인하였다.

• 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.

• Journal of Plant Biotechnology
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• 제7권4호
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• pp.219-223
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• 2005

We investigated chemical-wounding effect on Agrobacterium-mediated Chinese cabbage transformation via vacuum infiltration. Pre-germinated or germinating Chinese cabbage seeds were infiltrated with Agrobacterium tumefaciens LBA4404 cells carrying either GUS gene (pBI121) or hepatitis B virus surface antigen DNA (pBIHBsAg). Prior to agroinfiltration process, the seeds were soaked in sodium hydrosulfite (SHS) solution or just in sterile water as a control. Comparative transformation efficiency was determined by both of histochemistry and ELISA. We could demonstrate that SHS solution treatment especially to 1-day or 2-days old germinating seeds efficiently improved transformation process, and therefore, transient expression level. This strongly indicated that Agrobacterium infection could be facilitated indeed by SHS-causing wounds on Chinese cabbage seeds.