In vitro tissue culture and genetic analysis of two high-CBD medical cannabis (Cannabis sativa L.) breeding lines

Abstract

The species Cannabis sativa L. has recently witnessed a resurgence of interest all over the world due to its multipurpose applications and the scientific confirmation of its pharmacological properties. Genotypes with high cannabinoid content are appreciated in the pharmaceutical and cosmetic industries due to their therapeutic potential. These genotypes, with predominantly high cannabidiol (CBD) content, are the subject of research and breeding in several programs, but to date, little data is published on the in vitro tissue culture of cannabis. Our study focused on the establishment of an efficient micropropagation method for two high-CBD breeding lines (MX-CBD-11 and MX-CBD-707) as the basis for advanced biotechnological breeding approaches. The results demonstrated that the in vitro culture of medical cannabis can be initiated on different culture media, that cultured plants can be successfully acclimatized, and that nodal position, and especially the genotype, have a significant influence on the success of shoot culture establishment. They showed that the published tissue culture media optimized for one high-THC strain of Mexican cannabis are not as efficient for other genotypes of (medical) cannabis. We complemented this research with a genetic study of 95 plants of the two breeding lines with 16 microsatellite (SSR) markers which clustered the plants based on breeding line. The results demonstrated that only 8 markers are needed for discrimination of all analyzed plants and their usefulness for clonal identification.

The Past, Present and Future of Cannabis sativa Tissue Culture

“…In contrast to these reports of prolific MSCs, some authors have noted that Cannabis does not readily produce MSCs and instead tends to produce a single shoot with a high degree of apical dominance and low levels of branching, resulting in a much lower multiplication rate (Table 1) [75,78,80]. A recent study of two high-CBD Cannabis cultivars by Mestinšek Mubi et al [80] found shoot multiplication between 0.59 and 1.78 on TDZ or meta-Topolin (mT) media recipes, which had previously been reported to yield between 11 and 13 shoots by other research groups [72]. Furthermore, the authors found that these media compositions did not significantly improve shoot proliferation over the PGR-free control (MS) in their two tested cultivars [80].…”

“…A recent study of two high-CBD Cannabis cultivars by Mestinšek Mubi et al [80] found shoot multiplication between 0.59 and 1.78 on TDZ or meta-Topolin (mT) media recipes, which had previously been reported to yield between 11 and 13 shoots by other research groups [72]. Furthermore, the authors found that these media compositions did not significantly improve shoot proliferation over the PGR-free control (MS) in their two tested cultivars [80]. Similarly, Wróbel et al [78] report that existing nodal propagation protocols result in multiplication rates of 0.9, resulting in a loss of plant material.…”

“…As previously highlighted, some of the frequent challenges in Cannabis micropropagation include the lack of multiple shoot formation, a strong degree of apical dominance leading to low levels of branching, and poor survival of single-node explants. As a result, many studies using nodal tissues report low multiplication rates ranging from less than one (resulting in loss of stock plants) to about four explants per nodal culture (Table 1) [66,75,77,78,80]. From a practical standpoint, these multiplication rates are not suitable for many applications.…”

“…Reproducibility of existing methods remains a challenge in both regeneration [89] and SM systems [80]. As previously discussed, with most contemporary US-based studies presenting data from a single cultivar [28,78,85,86], it is likely that this lack of genotypic diversity is contributing to the struggle with protocol reproducibility in Cannabis tissue cultures.…”

“…No existing Cannabis micropropagation and regeneration studies have compared the effects of carbohydrate sources on regeneration or explant growth. Currently, sucrose remains the carbohydrate source of choice when preparing media for In Vitro studies of Cannabis [70,77,78,80,89]. Hence, it seems that studying the effects of carbohydrate sources on Cannabis micropropagation may be an avenue for improving available In Vitro regeneration systems.…”