Abstract:
Abstract
Recalcitrance to transformation makes genetic engineering of many valuable plants infeasible for practical use. Transitory host modification during transformation using transgenes offers a possible means of overcoming this obstacle. In prior work in our laboratory, the genes GA20ox7 and EBB1 were found to increase regeneration in vitro. In this project, I reanalyzed the regenerative properties of these two genes using a model poplar (hybrid Populus tremula x P. alba INRA 717-1B4) and a genotype that is recalcitrant to transform, P. trichocarpa (Nisqually-1):N-1. Three replicate transformation experiments were performed to compare callus, shoot, and transgenic shoot formation with the two genes and an Empty-Vector control. No shoots were produced by N-1 explants with or without the genes. 717 explants transformed with p409S:GA20ox7 regenerated poorly, producing a low proportion of transgenic shoots for stem (12.4%) and leaf (8.19%) explants compared to that for empty vector controls (24.4%, 29.5%). 717 explants transformed with p409S:EBB1, however, had a greater numbers of shoots per stem and leaf explant (37.7% and 54.2% increase, respectively) and a higher proportion (50.8%) of transgenic shoots from 717 leaf explants (44.5%) compared to the Empty-Vector controls (29.5% ). Additionally, callus growth on N-1 stem explants was increased by 116% by EBB1 and 76.8% by GA20ox7 compared to Empty-Vector controls. I conclude that the EBB1 transgene shows promise in improving transformation, but it must be removed or reactivated shortly after transformation due to apparent cytokinin toxicity and morphological defects.
