Transient expression of minimum linear gene cassettes in onion epidermal cells via direct transformation.
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 degrees 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.
Cheng
Yun-Qing
YQ
Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian, Liaoning, China.
Yang
Jun
J
Xu
Feng-Ping
FP
An
Li-Jia
LJ
Liu
Jian-Feng
JF
Chen
Zhi-Wen
ZW
D005822:Genetic Vectors / Q000235:genetics; D008853:Microscopy; D019697:Onions / Q000166:cytology* / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012231:Rhizobium / Q000235:genetics; D014169:Transformation, Bacterial