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

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 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.

Optimization of soybean (Glycine max (L.) Merrill) in planta ovary transformation using a linear minimal gus gene cassette

“…Fluorescein isothiocyanate (FITC)-labelled DNA tracing studies have shown that exogenous DNA could enter the soybean embryo sac more readily via a shortened pathway created by ovary-cutting . Direct transformation of a linear gene cassette in onion lower epidermal cells revealed that, although lacking the mediation of the Agrobacterium plasmid, the exogenous gene could enter the host cell nucleus and be successfully expressed (Cheng et al, 2009). The frequency of successful soybean ovary-drip transformation is about 3% .…”

Cloning and expression analysis of the R2R3-PsMYB1 gene associated with bud dormancy during chilling treatment in the tree peony (Paeonia suffruticosa)

“…The PsMYB1-GFP expression vector was used to transform onion epidermal cells according to the method of Cheng et al (2009). GFP fluorescence was then detected using an 80i Nikon fluorescent microscope under a stimulating wavelength of 488 nm.…”