| The primary objective of this research was to develop protocols for the Agrobacterium tumefaciens-mediated transformation of three elite hybrid aspen clones, Populus canescens × P. grandidentata (XCaG, Clone 36-67-S-1), P. tremuloides × P. tremula (XTTa, Clone 65-60-S-5), and P. tremuloides × P. davidiana (XTDa, Clone 18-59-S-11). Two disarmed A. tumefaciens strains (LBA 4404 and EHA 105, both with the pBI 121 binary vector) were employed. The T-DNA of pBI 121 contains an npt II gene conferring kanamycin resistance to select for transformed plant cells and the Escherichia coli uidA gene for β-glucuronidase to facilitate the visual identification of transformants via histochemical assay.; For all three hybrids, transgenic plants were regenerated and confirmed by DNA analysis. Transformation frequencies were generally highest when shoot regeneration was indirect, selection was immediate, and the leaves used as an explant source were harvested from younger shoot cultures (< 50 days old). For all three hybrids, transformation frequencies were improved significantly (typically two- to threefold) when the Agrobacterium cultures were pretreated with acetosyringone for virulence gene induction. When “physiologically older” leaves were employed, transformation frequencies were further improved by spiking the co-cultivation medium with acetosyringone.; On a per experimental run basis, mean transformation frequencies of 70 to 90% were achieved for the XCaG and XTTa hybrids, respectively, and multiple transgenic calli were typically regenerated from each leaf, however, the overall efficacy of the protocols was frequently limited at the shoot regeneration stage. For all three hybrids, most of the LBA 4404-derived callus lines were “proliferative” in phenotype and produced shoots only erratically if at all. Even when EHA 105 was employed, the harvestable shoot regeneration frequencies for the P. tremuloides hybrids never exceeded 50%. For the XTTa hybrid, the limitation appeared to be a consequence of the inclusion of carbenicillin for Agrobacterium elimination. Carbenicillin promoted hyperhydric (syn. waterlogged) shoot regeneration, however, its adverse effects could be largely counteracted by changes in nutrient salt formulation. Callus tissues from the XTDa hybrid were also susceptible to carbenicillin-mediated hyperhydricity, however, the small size of the XTDa calli may have also contributed to their recalcitrance. |
