• Journal of Plant Biotechnology
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• v.31 no.4
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• pp.279-283
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• 2004

To lily (Lilium longflorum cv. Georgia) pollen, impacts by some physical, chemical and biological factors were examined in respects of its growth and transient gene expression via agro-infiltration. Rolling movement in liquid medium or vacuum pressure during Agro-infiltration was regarded as a impact that should be minimized for normal pollen growth. Pollen growth was maintained well in relatively broad range of temperature (19 to 27$^{\circ}C$) or pH (5.0 to 8.0). Chemical factors such as cefotaxime (up to 300mg/L), acetosyringone (up to 800 $\mu$M) and syringealdehyde (up to 800 $\mu$M) did not show any harmful effects but kanamycin severely did even at concentration as low as 25mg/L in some cases. For GUS gene expression, acetosyringone at 200 to 400 $\mu$M slightly improved the efficiency while syringealdehyde did not. Brief agro-infiltration followed by 18 hr of co-incubation of pollen along with Agrobacterium was suggested as a condition basically required for the transient expression system using lily pollen regardless of the presence of acetosyringone.

• Plant Biotechnology Reports
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• v.5 no.3
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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.

• Journal of Plant Biotechnology
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• v.49 no.1
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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 agriculture & life science
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• v.45 no.6
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• pp.193-199
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• 2011

An efficient transient expression system has been developed and characterized for the production of foreign genes in seedlings. The seedlings can be easily produced from commercial seeds used for vegetable sprouts. In principal, a chemical abrasive was employed to generate wounds in seedlings prior to vacuum-infiltration with Agrobacterium tumefaciens bearing the target gene. This optimized chemical wounding-assisted agro-infiltration process resulted in up to 15-fold increase in $\beta$-glucuronidase (GUS) enzyme activity. This procedure has been used efficiently to express hepatitis B surface antigen (HBsAg) protein in a transient mode. Therefore, seedlings with proper wounds can be suggested as a convenient tool for the production of useful recombinant proteins.

• Journal of Plant Biotechnology
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• v.4 no.4
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• pp.171-178
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• 2002

Chlorophyll (Chl) fluorescence imaging was used to investigate the effectiveness of in vivo incorporation methods for two chemicals, 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) and methyl viologen (MV) in rice, a monocot, and cucumber, a dicot, leaves. four different methods (vacuum infiltration, floating, transpiration-aided incorporation through petiole and spraying) were compared, and $F_i$ and $F_v$/$F_m$ were chosen for the imaging of the DCMU- and MV-treated leaves, respectively. The effects of the chemicals in plants were generally heterogeneous over the whole leaf area. Moreover, the effectiveness of the treatment of a chemical in plant leaves was dependent on chemical species, plant species, concentration of the chemical, the treatment method, the duration of the treatment, the existence of light and detergent, etc. In conclusion, we suggest that to achieve the proposed effects of chemicals in plants for an actual experiment, these factors must be considered before the chemical treatment, and the best method for the treatment of the chemicals tested was floating and vacuum infiltration in the dicot and the monocot leaves, respectively, as drawn from Chl fluorescence imaging analysis.

• KSBB Journal
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• v.19 no.3
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• pp.174-177
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• 2004

A method of collection and long-term storage of viable lily (Lilium longiflorum) pollen grains were developed for their in vitro growth and transformation in consistency. Petroleum ether, n-heptane, cyclohexane and benzene, as pollen collection medium, were determined less toxic to pollen growth in vitro than others tested. Pollen grains, however, lost their growth activity if stored in these solvents more than a week, So, a serial performance, that is, pollen grain collection in these solvents, air-drying and immediate transfer to low temperature condition was determined desirable for keeping the viability much longer. Pollen grains from this storage showed a successful transformation in vitro with a cDNA encoding tissue plasminogen activator (TPA) protein using Agrobacterium via vacuum infiltration according to western blotting analysis.

• KSBB Journal
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• v.17 no.6
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• pp.577-581
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• 2002

In an attempt to produce recombinant proteins using the pollen enriched in some plant species, a 1.7 kb DNA encoding urease subunit B (UreB) amplified by PCR from Helicobacter pylori urease gene cluster in pH808 plasmid was cloned to be expressed under CaMV35S promoter in lily (Lilium longiflorum) pollen tubes elongated in vitro. Lily pollen at early germinating stage was transformed with the ureB DNA using Agrobacterium via vacuum infiltration and, incubated for a full pollen tube growth 16 - 24 h in the dark in the presence of kanamycin. DNA integration and expression in the transgenic pollen were analyzed by the standard molecular techniques and the results suggest that the pollen in vitro may be employed as a protein factory in a disposable fashion.

• KSBB Journal
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• v.23 no.6
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• pp.554-556
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• 2008

Fresh Oenanthe javanica DC. leaves still attached to stem architecture were immersed in NaOH solution for 3 min before agroinfiltration and co-cultivation. MTT assay revealed that NaOH solution containing up to 0.7% was still safe for the leaf viability. Fluorometric GUS enzyme analysis showed that 0.5% NaOH-treated leaf tissues were efficiently transformed by vacuum infiltration for 20 min with Agrobacterium cells at a density of $OD_{600}=0.5$ to 1.0. These conditions worked well for the expression of HBsAg, which was confirmed by western blotting and ELISA.

• Journal of Plant Biotechnology
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• v.48 no.3
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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.

• Nuclear Engineering and Technology
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• v.56 no.6
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• pp.2367-2374
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• 2024

In this paper, based on the need for high-strength connections between all-tungsten-oriented plasma materials and thermal sinking materials of copper and its alloys in nuclear fusion devices, a study on the effect of tungsten surface laser micro structuring on the interfacial bonding properties of W/Cu joints was carried out. In the experiment, the connectors were prepared by vacuum fusion infiltration technology, and the effects of microgroove structure on the mechanical and thermal conductivity of W/Cu connectors were investigated under different parameters (including microgroove pitch, microgroove depth, and microgroove taper). The maximum shear strength is 126.0 MPa when the pitch is 0.15 mm and the depth is 34 ㎛. In addition, the negative taper structure, i.e., the width of the entrance of the microstructure is smaller than the width of the interior of the microstructure, is also investigated. The shear tests show that there is an approximately linear relationship between the shear strength of W/Cu and taper. Compared with the positive taper, the shear strength of the samples with the same morphological density and depth of the tungsten surface is significantly higher.