|Abstract or Summary
- Celiac disease, an autoimmune response triggered by the consumption of seed storage proteins of cereals, affects an estimated 1% of the human population. In the case of wheat (Triticum aestivum L.), one of the world’s most consumed cereals, alcohol-soluble proteins, the gliadins, have been identified as the allergy-eliciting agents. Previous studies have shown that removal of gliadins may be a feasible method of producing non-toxic wheat food preparations. Thus, the aim of this study was to explore a transgenic-based method using a mechanism of post-transcriptional gene regulation, RNA interference (RNAi), to decrease the levels of wheat gliadins thereby decreasing the amount of the allergy-eliciting activity in its flour. The aim of this project was addressed in two phases. The first involved the optimization of a biolistics-based wheat transformation system and the isolation of transgenic lines. The second phase involved phenotypic evaluations of transgenic lines to determine the effectiveness of RNAi on decreasing gliadin levels and related seed storage proteins. For the first phase of this project, optimization of wheat transformation parameters using the BioRad PDS-1000/He biolistics gene gun with the Hepta adapter was accomplished by testing the effect of microprojectile size, amount of microprojectiles, and the bombardment pressure on transformation success. Additionally, the utility of a transiently expressed reporter coding for the green fluorescent protein (GFP) in predicting the rate of transformation success two days post-bombardment was examined. The pMCG161|irGLI (pGLI) binary vector containing the bar gene (conferring glufosinate resistance) and a gliadin inverted repeat (RNAi component) was co-bombarded with the pAHC17|sGFP-SK (pGFP) vector containing a GFP reporter gene in a 1:1 mass ratio. Using a factorial design, the size of the microprojectile was statistically significant (p = 0.001) in transformation success. A significantly higher (>6x) success rate using particles with a diameter of 0.6-µm over the use of 1.0-µm microprojectiles was noted. The interaction between bombardment pressure and microprojectile size was also significant with pressures of 1350 and 1100 PSI being superior to 1550 PSI when 0.6-µm microprojectiles were used. Using a bombardment pressure of 1350 PSI and 0.6-µm particles, the best transformation rate of 2.5% was achieved. The amount of microprojectiles per microcarrier (0.14 or 0.5 mg) did not significantly affect transformation success. Scoring transient GFP expression two days post-bombardment was not found to be a useful predictor of transformation success. For the second phase of this project, regenerated plants were evaluated for expression of the bar gene selectable marker (that conferred resistance to the glufosinate herbicide) and gliadin protein composition using reverse phase high pressure liquid chromatography (RP-HPLC). Of the 11,962 immature embryos bombarded with pGLI and pGFP, 189 independently transformed lines were recovered with 102 lines showing resistance to glufosinate, a sign of an actively expressed bar gene introduced with pGLI. Protein analysis on 117 transformants (69 T1 and 48 T2 individuals derived from 13 initial T0 transgenics) did not reveal qualitative differences in gliadin levels or composition between transgenics and the untransformed control suggesting that the RNAi component of pGLI had failed to induce silencing.