Basal media optimization for the micropropagation and callogenesis of Cannabis sativa L. S.R.G. Page, A.S. Monthony, and A.M.P. Jones Gosling Research Institute for Plant Preservation, Department of Plant Agriculture, University of Guelph, Guelph, N1G 2W1, ON, CANADA Abstract Micropropagation of Cannabis sativa L. is an emerging area of research for genetic storage and large-scale production of clean planting material. However, existing protocols were developed using a limited number of genotypes and are often not reproducible. Previous studies reported MS + 0.5 M TDZ to be optimal for Cannabis micropropagation, yet in our preliminary studies this medium resulted in excessive callus formation, hyperhydricity, low multiplication, and high mortality rates. Following an initial screen of five basal salt mixtures commonly used for micropropagation (WPM, MS, B5, BABI, and DKW), we determined that DKW produced the healthiest plants. In a second experiment, the multiplication rate and canopy area of explants grown on MS + 0.5 M TDZ and DKW + 0.5 M TDZ were compared using five drug-type cultivars to determine if the preference for DKW was genotype-dependent. Four out of five genotypes had significantly higher multiplication rates on DKW + 0.5 M TDZ with the combined average being 1.5x higher than explants grown on MS + 0.5 M TDZ. The canopy area was also significantly larger for plants cultured on DKW + 0.5 M TDZ for four out of five genotypes with the combined average being twice that of explants grown on MS + 0.5 M TDZ. In the third experiment, callogenesis was compared using a range of 2,4-D concentrations (0-30 M) on both MS and DKW. Greater callus growth was observed on DKW than on MS. While further improvements are likely possible through media optimization, this study represents an important step towards developing standardized micropropagation practices for Cannabis. . CC-BY-NC-ND 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted February 9, 2020. . https://doi.org/10.1101/2020.02.07.939181 doi: bioRxiv preprint