Green fluorescent protein as a reporter gene for orchardgrass leaf transformation

The green fluorescent protein (GFP) genes have become preferred reporter genes for transformation experiments because the assay is nondestructive and gene expression can be quickly screened under a light microscope without the addition of exogenous cofactors or extensive detection equipment. The overall objective of this project was to examine the potential application of GFP as a reporter for transformation of orchardgrass (Dactylis glomerate L.) leaf cells.

The innermost leaves from orchardgrass tillers were split in half along the midvein, the basal 16 mm cut transversely into 4 mm x 4 mm segments, and plated on Schenk and Hildebrandt (SH) medium. The leaf segments were transferred to SH medium containing 150 gL^-1 sucrose 4 h before bombardment as an osmotic treatment. Tungsten particles, coated with plasmid DMA, were delivered via a particle inflow gun.

The first goal was to identify which of four promoters; maize (Zea mays L.) ubiquitin (ubi1), cauliflower mosaic virus (CaMVSSS), rice (Oryza sativa L.) actin (act1), or rice rubisco (rbcS), resulted in the highest level of gene expression. The uidA reporter gene (GUS) was used to quantify the strength of gene expression by each promoter. GUS is an Escherichia coli gene which codes for an enzyme that hydrolyzes β-glucuronide substrates which subsequently react with oxygen to form a blue precipitate. The bombarded segments were treated with substrate, and the mean number of blue spots per leaf segment was determined. Gene expression driven by ubi1 and act1 promoters resulted in the highest level of GUS expression. The CaMV35S promoter was significantly weaker than both, and the rbcS promoter did not result in any GUS expression.

There are several different GFP coding regions that can be used for plant transformation experiments. The second goal of this project was to compare three of these genes, the wild-type GFP, mGFP, and sGFP, to determine which yielded the highest level of fluorescence. The mGFP gene is a version modified by site-directed mutagenesis to alter the sequence of a cryptic intron. This intron produces a site on the transcript that is misspliced in Arabidopsis thaliana cells. The sGFP gene is engineered with an alternate nucleotide coding sequence that results in brighter fluorescence in maize (Zea mays L.). Several GFP gene and promoter combinations were compared including GFP and mGFP driven by CaMV35S, GFP driven by ubi1, and sGFP driven by act1. GFP and mGFP did not produce detectable fluorescence, but the sGFP construct did result in gene expression.

The third goal of this project was to insert a GFP gene into a plasmid that contains a selectable marker gene. The sGFP gene cassette was added to a vector containing the bar gene, which confers tolerance to phosphinothricin based herbicides. This construct, psGFP-BAR, was used in transformation experiments and transient GFP expression was detected. The combination of sGFP and bar gives a nondestructive reporter and a selectable marker for use in future transformation experiments.