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Transient Expression of Minimum Linear Gene Cassettes in Onion Epidermal Cells Via Direct Transformation

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Abstract

A new method without any special devices for direct transformation of linear gene cassettes was developed. Its feasibility was verified through 5′-fluorescent dye (fluorescein isothiocyanate, FITC)-labeled fluorescent tracing and transient expression of a gus reporter gene. Minimal linear gene cassettes, containing necessary regulation elements and a gus reporter gene, was prepared by polymerase chain reaction and dissolved in transformation buffer solution to 100 ng/mL. The basic transformation solution used was Murashige and Skoog basal salt mixture (MS) liquid medium. Hypertonic pretreatment of explants and transformation cofactors, including Ca2+, surfactant assistants, Agrobacterium LBA4404 cell culture on transformation efficiency were evaluated. Prior to the incubation of the explants and target linear cassette in each designed transformation solution for 3 h, the onion low epidermal explants were pre-cultured in darkness at 27 °C for 48 h and then transferred to MS solid media for 72 h. FITC-labeled linear DNA was used to trace the delivery of DNA entry into the cell and the nuclei. By GUS staining and flow-cytometry-mediated fluorescent detection, a significant increase of the ratios of fluorescent nuclei as well as expression of the gus reporter gene was observed by each designed transformation solution. This potent and feasible method showed prospective applications in plant transgenic research.

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Acknowledgments

The authors acknowledge the financial support provided by the National R&D Project of Transgenic Crops of Ministry of Science and Technology of China (JY03-B-18-02), Dalian Municipal Science and Technology Program (2007B10NC137) and Key Laboratory of Industrial Ecology and Environmental Engineering (Dalian).

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Authors and Affiliations

  1. Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian, 116024, Liaoning, China

    Yun-Qing Cheng, Jun Yang, Feng-Ping Xu & Li-Jia An

  2. School of Life Science, Jilin Normal University, Siping, 136000, Jilin, China

    Jian-Feng Liu & Zhi-Wen Chen

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  1. Yun-Qing Cheng
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  2. Jun Yang
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  3. Feng-Ping Xu
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  4. Li-Jia An
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Correspondence to Jun Yang.

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Cheng, YQ., Yang, J., Xu, FP. et al. Transient Expression of Minimum Linear Gene Cassettes in Onion Epidermal Cells Via Direct Transformation. Appl Biochem Biotechnol 159, 739–749 (2009). https://doi.org/10.1007/s12010-009-8554-7

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  • DOI: https://doi.org/10.1007/s12010-009-8554-7

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