Micropropagation is a valuable production tool for the cultivation of hemp (Cannabis sativa), and development of optimal protocols is ongoing. The goal of this study was to evaluate a novel growing medium combination, consisting of Driver and Kuniyuki Walnut (DKW) medium as the nutrient source and glucose as the carbon source, and to investigate the link between in vitro and in vivo (i.e., greenhouse) plant performance. Among 10 accessions intended to represent a range of heterozygosity levels and various essential oil chemotypes, the DKW–glucose growing medium generally produced the most vigorous plantlets by all parameters evaluated in vitro (height, biomass, canopy area, vegetative growth rate, and regeneration rate). Across four growing media treatments, all of which included meta-topolin as the sole plant growth regulator, poor to no rooting was observed in vitro. Hybrids were more vigorous than nonhybrid selections in vitro, but not in vivo. No correlation was observed between in vitro and in vivo vigor, indicating that, with these media, plant performance in vitro is not predictive of that in vivo.
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