• 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.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2018.04a
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• pp.36-36
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• 2018

Soybean is the important crop in Asian countries as protein source, oil production and animal feed. Improving soybean using genetic transformation is the principal tool in nowadays. Developing herbicide resistant transgenic soybean plants through Agrobacterium-mediated transformation has been worked in many previous studied. However, the transformation efficiency is still low. Many attempts try to find the optimum media condition for plant regeneration after infection. After transformation, the plant regeneration is very important condition to promote growth of transgenic plant. In this study, we optimized a regeneration condition for two Korean soybean cultivar, Dawonkong and Pungsannamulkong using cotyledon, cotyledonary nodes and hypocotyl as explant. The results showed that shoot regeneration of cotyledonary nodes on B5 medium containing 2 mg/L 6-benzylaminopurine showed the highest percentage of regeneration in Dawonkong (75.8%) while Pungsannamulkong presented high number of shoots 2.12 shoots per explant. For transformation condition, co-cultivation in 7 days showed a high number of GUS positive expression. Most of explants can survived under media including 5 mg/L of glufocinate which refers phosphinotricin for 2-week selection. Washing with 400 mg/L of cefotaxime in several times and selection in plant regeneration media with 400 mg/L of cefotaxime can prevent bacteria growth, effectively.

• Journal of Crop Science and Biotechnology
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• v.10 no.2
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• pp.92-97
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• 2007

Antibiotics used for suppressing Agrobacterium in plant transformation procedure might have negligible effects on plant tissues and regeneration. The effects of antibiotics on growth suppression of Agrobacterium and plant regeneration were investigated for enhancing Agrobacterium-mediated transformation using wheat mature embryos. Antibiotics tested, except carbenicillin, were able to suppress that embryos were coated with a layer of Agrobacterium cells in callus induction medium. Agrobacterium growth was suppressed minimally at 50 mg/l of timentin, while cefotaxime and clavamox were completely suppressed at relative high concentration of 250 mg/l. In the treatment of carbenicillin, initiation of growth suppression of Agrobacterium occurred at 750 mg/l of concentration because Agrobacterium KYRT1 contains the carbenicillin resistant gene. In Agrobacterium inoculation, effects of antibiotics were significantly different on the rate of callus induction and shoot formation. Almost embryos were induced calli at 50 mg/l of timentin whereas callus induction rate was achieved above 90% at 100 mg/l and 250 mg/l of cefotaxime and clavamox, respectively. Shoot formation rate was higher in the treatment of timentin than that of cefotaxime and clavamox at 500 mg/l of concentration, respectively. Timentin can be used as a good antibiotics in Agrobacterium-mediated wheat transformation.

• Plant Resources
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• v.7 no.1
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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.

• Mycobiology
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• v.45 no.2
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• pp.84-89
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• 2017

Fungi of the Metarhizium genus are a very versatile model for understanding pathogenicity in insects and their symbiotic relationship with plants. To establish a co-transformation system for the transformation of multiple M. robertsii genes using Agrobacterium tumefaciens, we evaluated whether the antibiotic nourseothricin has the same marker selection efficiency as phosphinothricin using separate vectors. Subsequently, in the two vectors containing the nourseothricin and phosphinothricin resistance cassettes were inserted eGFP and mCherry expression cassettes, respectively. These new vectors were then introduced independently into A. tumefaciens and used to transform M. robertsii either in independent events or in one single co-transformation event using an equimolar mixture of A. tumefaciens cultures. The number of transformants obtained by co-transformation was similar to that obtained by the individual transformation events. This method provides an additional strategy for the simultaneous insertion of multiple genes into M. robertsii.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.49 no.5
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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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• v.31 no.1
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• pp.13-17
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• 2004

To improve transformation efficiency, the callus of Lilium lancifolium Thunb. were bombarded by particles coated with pIG 121 Hm which include NPT II and GUS genes, and then cocultivated with Agrobacterium tumefaciens EHA101 which contain pIG121Hm binary vector, carrying neomycin phosphotransferase (NPT II) and $\beta$-Glucuronidase (GUS) genes. Three days after cocultivation with Agrobacterium tumefaciens and particle bombardment, the callus clusters were transferred to MS medium containing 1mg/L 2,4-D, 0.1mg/L BAP, 100mg/L kanamycin and 200mg/L carbenicillin. Four weeks after transfer to the selection medium, GUS expression was detected and PCR analysis revealed the presence of NPT II fragment of the expected size (700 bp) in the transformed callus. The GUS expression from Agrobacterium-mediated transformants after particle bombardment increased to over 3-folds compared with that of callus cocultivated with Agrobacterium tumefaciens without particle bombardment. The callus clusters containing kanamycin resistant gene were transferred to MS medium containing 1mg/L NAA and 1mg/L BAP. Somatic embryos were developed in four weeks and microbulbs expressing GUS were formed.

• Mycobiology
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• v.43 no.1
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• pp.1-8
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• 2015

Mushroom transformation requires a series of experimental steps, including generation of host strains with a desirable selective marker, design of vector DNA, removal of host cell wall, introduction of foreign DNA across the cell membrane, and integration into host genomic DNA or maintenance of an autonomous vector DNA inside the host cell. This review introduces limitations and obstacles related to transformation technologies along with possible solutions. Current methods for cell wall removal and cell membrane permeabilization are summarized together with details of two popular technologies, Agrobacterium tumefaciens-mediated transformation and restriction enzyme-mediated integration.

• Journal of Plant Biotechnology
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• v.6 no.2
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• pp.69-75
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• 2004

Optimal protocol for efficient genetic transformation has been defined to aid future strategies of genetic engineering in pigeon pea with agronomically important genes. Transgenic pigeonpea plants were successfully produced through Agrobacterium tumefaciens-mediated genetic transformation method using cotyledonary node explants by employing defined culture media. The explants were co-cultivated with A. tumefaciens strain C-58 harboring the binary plasmid, pCAMBIA-1301 [con-ferring $\beta$-glucuronidase(GUS) activity and resistance to hygromycin] and cultured on selection medium (regeneration medium supplemented with hygromycin) to select putatively transformed shoots. The shoots were then rooted on root induction medium and transferred to pots containing sand and soil mixture in the ratio of 1:1. About 22 putative TO transgenic plants have been produced. Stable expression and integration of the transgenes in the putative transgenics were confirmed by GUS assay, PCR and Southern blot hybridization with a transformation efficiency of over 45%. Stable integration and expression of the marker gene has been confirmed in the TO and T1 transgenics through PCR, and Southern hybridization.

• Journal of Microbiology and Biotechnology
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• v.18 no.4
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• pp.754-758
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• 2008

Agrobacterium tumefaciens-mediated transformation (ATMT) was successfully applied to Monascus ruber. The optimum cocultivation time was 84 h with an efficiency of 900 to 1,000 transformants when $1{\times}10^6$ spores were used with the same volume of bacteria. The stability of transform ants was over 98% after five generations. When M. ruber was transformed with A. tumefaciens YL-63 containing the green fluorescent protein gene (egfp), the green fluorescent signal was observed throughout hyphae, confirming expression of the gene. This efficient transformation and expression system of M. ruber by ATMT will facilitate the study of this fungus at a molecular genetic level.