Toby Cicak

Image Analysis of Transient Expression in Bombarded Soybean (Glycine max) Immature Embryos.

Particle bombardment of cells traditionally results in low frequencies of regenerated transgenic plants, which could be due to low (1-2%) cell viability in particle containing cells (Hunold, Bronner, & Hahne, 1994). Several methods for reducing damage to cells during bombardment have been developed, including the use of osmotic treatments (Vain, McMullen, & Finer, 1993) and the reduction of gold particle size (Randolph-Anderson, et al., 1995). It was determined that reducing the total number of gold particles per shot at the target tissue might reduce the damage received by each cell, thereby improving cell survival and regeneration. Immature embryos of soybean were bombarded using five different concentrations of gold particles to determine the effects of gold particles as measured by transient expression. Image analysis of bombarded immature embryos was performed using ImageJ to determine the number and location of foci with the bombardment target area and this information was used to optimize concentration of gold density for particle bombardment.

Expression of a novel bi-directional Brassica napus promoter in soybean

The expression profile of a natural bi-directional promoter, derived from the Brassica napus EPSPS-A gene, was studied in transgenic soybean (Glycine max C.V. Maverick) lines. Two constructs, pDAB100331 and pDAB100333, were assembled to test the bi-directionality of the promoter. Two reporter genes, gfp and gusA, were employed and they were interchangeably placed in both constructs, one on each end of the promoter such that both proteins expressed divergently in each construct. In the T0 generation, GUS expression was more uniform throughout the leaf of pDAB100333 transgenic plants, where the gusA gene was expressed from the downstream or EPSPS-A end of the bi-directional promoter. Comparatively, GUS expression was more localized in the midrib and veins of the leaf of pDAB100331 transgenic plants, where the gusA gene was expressed from the upstream end of the bi-directional promoter. These observations indicated a unique expression pattern from each end of the promoter and consistently higher expression in genes expressed from the downstream end (e.g., EPSPS-A end) of the promoter in the tissues examined. The GFP expression pattern followed that of GUS when placed in the same position relative to the promoter. In the T1 generation, transcript analysis also showed higher expression of both gusA and gfp when those genes were located at the downstream end of the promoter. Accordingly, the pDAB100331 events exhibited a higher gfp/gusA transcript ratio, while pDAB100333 events produced a higher gusA/gfp transcript ratio consistent with the observations in T0 plants. These results demonstrated that the EPSPS-A gene bidirectional promoter can be effectively utilized to drive expression of two transgenes for the desired traits.