DKW basal salts improve micropropagation and callogenesis compared with MS basal salts in multiple commercial cultivars ofCannabis sativa

Abstract

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.

Recalcitrance of Cannabis sativa to de novo regeneration; a multi-genotype replication study

“…As such, it can be concluded that either this method is not suitable for inducing de novo regeneration across different genotypes, or that one or more of the details that could not be accurately replicated (Table 1) are critical for success. Regardless, these data highlight previous observations that Cannabis is a relatively recalcitrant species [6,9,13,22,32] and care must be taken to include multiple genotypes and detailed descriptions of experimental designs to ensure reproducible methods.…”

“…All leaf material was sourced from in vitro shoots maintained within a long-term in vitro germplasm at the University of Guelph. All explants used in this experiment were from Stage 2 in vitro grown shoots maintained as previously described [6,13]. The plants had been in a state of vegetative growth for at least 6 months and were routinely subcultured ever 4-6 weeks.…”

“…Often the most successful methods have used the genotype MX from the University of Mississippi [7,[10][11][12]. Recent evidence suggests that micropropagation protocols developed using a single genotype of C. sativa may not be replicable when tested on other genotypes, instead resulting in significantly lower multiplication rates [13]. Notably, most micropropagation protocols published in the United States are developed with Cannabis supplied by the University of Mississippi, who is funded through the National Institute on Drug Abuse (NIDA) as the sole licensed facility to supply Cannabis for research purposes in the United States [14,15].…”

“…As the regulatory landscape evolves to facilitate research in this nascent field, there is growing interest in Cannabis regeneration (Fig 1) and an increasing need for reliable methods. Despite growing interest, the number of drug-type genotypes used in protocol development has historically been very low [5,12,21,28] and only recently have five or more genotypes been included in a single study [13,29]. In stark contrast, regeneration studies on commercial hemp varieties can boast up to 12 genotypes and has identified significant genotypic variability [18].…”

“…Generalizing conclusions based on single-genotype studies has faced scrutiny, and this is particularly poignant given the chemical and genetic differences identified between the genotypes used for research purposes and commercially relevant germplasm [14][15][16]. Despite growing evidence of the diversity of Cannabis, there have been limited in vitro attempts to assess the effects of this genetic diversity on replicability of existing tissue culture methods [13]. The objective of this study was to replicate the Lata et al, 2010 [11] methods as closely as possible across 10 different genotypes to assess the reproducibility of this protocol and to provide insight into the variability in genotypic response.…”