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

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 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.

Recalcitrance ofCannabis sativatode novoregeneration; a multi-genotype replication study

“…Work by Page et al . [9] has shown that the use of DKW media [28] may promote increased levels of callogenesis in some Cannabis genotypes, suggesting that media composition and genotype may interact to affect the formation of callus and subsequent regeneration potential.…”

“…The most successful protocols report between 9 and 13 shoot explants per nodal segment [4,7]. Unfortunately, most existing protocols were developed using a single high-THC genotype of C. sativa , and recent evidence suggests that methods developed using a single genotype are not replicable when tested on commercially available drug-type C. sativa genotypes and that multiplication rates are much lower, around 2.2 shoots per explant [9]. In addition to low regeneration rates, MSC is unsuitable for some biotechnological applications, and cannot compete with the relative ease of traditional vegetative propagation methods for large-scale commercial plant propagation.…”

“…In stark contrast, regeneration studies on commercial hemp varieties can boast up to 12 genotypes [11]. Generalizing conclusions based on single-genotype studies have faced scrutiny as mounting evidence shows Cannabis responds in a genotype specific manner in vitro , yet few replication studies have been conducted [9,23,24]. The objective of this study was to replicate the Lata et al ., 2010 methods across 10 different genotypes to assess the reproducibility of this protocol and the variability in genotypic response.…”

Photoperiodic Response of In Vitro Cannabis sativa Plants

“…Media composition. A previously optimized semi-solid tissue culture medium (Page et al, 2020) that consisted of 5.32 g/L DKW basal salts and vitamins (D2470; PhytoTech Laboratories, Shawnee, KS) (Driver and Kuniyuki, 1984), 30 g/L sucrose, 1 mL/L plant preservative mixture (PPM) (Plant Cell Technology, Washington, DC), and 6 g/L of agar (A360-500; Fisher Chemical, Fair Lawn, NJ) adjusted to a pH of 5.7 before being autoclaved was used. Approximately 200 mL of the medium was poured into the bottom of each sterile We-V tissue culture vessel.…”

“…In vitro flowering and seed set have been proposed in other species as a valuable tool to reduce generation time for applications in plant breeding and to study floral/seed development in a highly controlled environment (Ochatt et al, 2000). Additional aspects specific to cannabis may include the opportunity to study the regulation of secondary metabolites, production of floral tissue for plant regeneration (Piunno et al, 2019), and the potential to rapidly identify the critical photoperiod of specific genotypes.…”