The viability gambit: an optimized disinfection protocol for industrial hemp (Cannabis sativa L.) seeds

Hemp (Cannabis sativa L.) is an increasingly important crop with applications spanning fiber, seed oil and bioactive cannabinoid production, yet the development of reliable tissue culture systems for this species remains a significant challenge. The establishment of axenic seedling cultures is a prerequisite for hypocotyl-based regeneration and future genetic transformation pipelines, but hemp seeds can harbor diverse microbial communities which can make seed disinfection challenging. Here, we evaluate several disinfection strategies across seven industrial hemp cultivars including treatment with hydrogen peroxide and sodium hypochlorite as well as the Prochloraz-based fungicide Octave and Plant Preservative Mixture (PPM™) on both, in-house produced and commercially sourced seed material. Across all treatments and accessions, in-house glasshouse-harvested seeds achieved higher disinfection rates than commercially sourced material regardless of the protocol applied (n=3681 and 1886, respectively). Among the disinfection treatments evaluated, a baseline of 75% ethanol combined with sequential hydrogen peroxide or sodium hypochlorite incubation performed well for certain seed batches, though variation in viable seedling rate remained high (~30% to ~80% viable seedlings). Supplementation with PPM™ increased the rates of viable seedlings to ~60% in some cases. Beyond their effect on contamination, disinfection treatments appeared to influence the morphogenic fate of hypocotyl explants independently of disinfection outcomes. Here, seedling treatment with Octave was associated with shoot and root co-regeneration in the absence of exogenous plant growth regulators. Together, these findings provide a practical framework for axenic hemp seed culture that can be used as starting point requiring local adaptation based on seed source, batch history and the intended downstream application.