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

Content Provider	Springer Nature Link
Author	Cheng, Yun Qing Yang, Jun Xu, Feng Ping An, Li Jia Liu, Jian Feng Chen, Zhi Wen
Copyright Year	2009
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 Ca$^{2+}$, 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.
Starting Page	739
Ending Page	749
Page Count	11
File Format	PDF
ISSN	02732289
Journal	Applied Biochemistry and Biotechnology
Volume Number	159
Issue Number	3
e-ISSN	15590291
Language	English
Publisher	Humana Press Inc
Publisher Date	2009-03-03
Publisher Place	New York
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Minimum linear gene cassettes Onion low epidermal Transient gene expression Gus gene Transformation buffer Cofactors Biochemistry Biotechnology
Content Type	Text
Resource Type	Article
Subject	Medicine Molecular Biology Environmental Engineering Biochemistry Bioengineering Applied Microbiology and Biotechnology Biotechnology

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