AbstractIn preparation for a major GWAS (Genome Wide Association Study) of plant regeneration and transformation, a large number of factors were examined for their effects on indirect regeneration rate in diverse wild genotypes—seeking a high rate of regeneration, but also highly genetically variable and heritable treatments. Many of the factors examined have never before been reported on for their effects on callus, shoot, or root organogenesis in poplar (Populus). Stems had the highest regeneration potential, followed by petioles and leaves, with greenhouse grown explant sources superior to in vitro growth explant sources. Changes of ± 50% to Murashige and Skoog (MS) basal medium salts and micronutrients had a minor effect on regeneration. Many popular treatments that were evaluated also had little to no useful effect at the levels studied, including activated charcoal, ascorbic acid, silver nitrate, melatonin, serotonin, sucrose concentration, and lipoic acid. As a result of this wide exploration, treatment combinations that substantially elevated regeneration in diverse genotypes were identified, enabling GWAS.