The recent legalization of Cannabis sativa L. in many regions has revealed a need for effective propagation and biotechnologies for the species. Micropropagation affords researchers and producers methods to rapidly propagate insect-/disease-/virus-free clonal plants and store germplasm and forms the basis for other biotechnologies. Despite this need, research in the area is limited due to the long history of prohibitions and restrictions. Existing literature has multiple limitations: many publications use hemp as a proxy for drug-type Cannabis when it is well established that there is significant genotype specificity; studies using drug-type cultivars are predominantly optimized using a single cultivar; most protocols have not been replicated by independent groups, and some attempts demonstrate a lack of reproducibility across genotypes. Due to culture decline and other problems, the multiplication phase of micropropagation (Stage 2) has not been fully developed in many reports. This review will provide a brief background on the history and botany of Cannabis as well as a comprehensive and critical summary of Cannabis tissue culture. Special attention will be paid to current challenges faced by researchers, the limitations of existing Cannabis micropropagation studies, and recent developments and future directions of Cannabis tissue culture technologies.

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 μ 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 μ M TDZ and DKW + 0.5 μ 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 μ M TDZ with the combined average being 1.5x higher than explants grown on MS + 0.5 μ M TDZ. The canopy area was also significantly larger for plants cultured on DKW + 0.5 μ M TDZ for four out of five genotypes with the combined average being twice that of explants grown on MS + 0.5 μ M TDZ. In the third experiment, callogenesis was compared using a range of 2,4-D concentrations (0-30 μ 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.

Existing Cannabis sativa micropropagation protocols use a limited number of cultivars and are often not reproducible. Currently, MS + 0.5 μM TDZ has been reported as the optimal medium for nodal micropropagation, yet our preliminary studies with this medium have resulted in abnormal morphology and high mortality rates in multiple cultivars. Following an initial screen of basal salt mixtures (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 μM TDZ and DKW + 0.5 μM TDZ were compared using five drug-type cultivars. The combined multiplication average of explants grown on DKW + 0.5 μM TDZ was 1.5x higher than explants grown on MS + 0.5 μM TDZ. Similarly, the combined average of the canopy area was twice as large on DKW + 0.5 μM TDZ. In the third experiment, callogenesis was compared using a range of 2,4-D concentrations (0-30 μM) on both MS and DKW and similarly, callus growth was superior on DKW. This study presents the largest comparison of basal salt compositions on the micropropagation of five commercially grown Cannabis cultivars to date.

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Cannabis sativa is relatively recalcitrant to de novo regeneration, but several studies have reported shoot organogenesis or somatic embryogenesis from non-meristematic tissues. Most report infrequent regeneration rates from these tissues, but a landmark publication from 2010 achieved regeneration from leaf explants with a 96% response rate, producing an average of 12.3 shoots per explant in a single drug-type accession. Despite the importance regeneration plays in plant biotechnology and the renewed interest in this crop the aforementioned protocol has not been used in subsequent papers in the decade since it was published, raising concerns over its reproducibility. Here we attempted to replicate this important Cannabis regeneration study and expand the original scope of the study by testing it across 10 drug-type C. sativa genotypes to assess genotypic variation. In our study, callus was induced in all 10 genotypes but callus growth and appearance substantially differed among cultivars, with the most responsive genotype producing 6-fold more callus than the least responsive. The shoot induction medium failed to induce shoot organogenesis in any of the 10 cultivars tested, instead resulting in necrosis of the calli. The findings of this replication study raise concerns about the replicability of existing methods. However, some details of the protocol could not be replicated due to missing details in the original paper and regulatory issues, which could have impacted the outcome. These results highlight the importance of using multiple genotypes in such studies and providing detailed methods to facilitate replication.