1,209 genotypes of Populus trichocarpa underwent an in vitro transformation and regeneration protocol and were imaged with RGB and hyperspectral cameras (via macoPhor Array, Middleton Spectral Vision). Fluorescent-hyperspectral images enable the visualization of enhanced GFP (eGFP) used as a reporter for successful transformation. This dataset contains 19,961 Petri dish images, spanning three timepoints (after 3 weeks of callus induction, after a subsequent 4 weeks of shoot induction, and again after another 3 weeks of shoot induction). Each genotype is represented by a median of two replicates each of samples with and without an added acetosyringone pre-treatment. The GMOdetector system (https://github.com/naglemi/GMOnotebook) was used to obtain segmentation maps of regenerating tissues (e.g., callus, shoot) from RGB images and cross-reference these with reporter protein signals from fluorescent-hyperspectral images, ultimately producing statistics on regeneration and transformation frequencies of specific tissues. These statistics were then analyzed in a downstream genome-wide association study (GWAS) and tests for genetic interactions among GWAS candidates.