The Past, Present and Future of Cannabis sativa Tissue Culture

Abstract

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.

Advances and Perspectives in Tissue Culture and Genetic Engineering of Cannabis

“…Micropropagation is the first and foremost application of in vitro culture in cannabis [ 10 , 47 , 60 , 61 ]. While micropropagation for applications in genetic preservation or propagation are generally achieved through shoot proliferation from existing meristems, many applications in biotechnology require the establishment of de novo regeneration in which plants are produced from non-meristematic tissues.…”

“…However, the optimal condition for in vitro propagation may not be optimal to preserve the genetic integrity of the regenerated genotype [ 151 ]. Indeed, in vitro conditions such as medium composition, PGRs, high humidity, the number of subcultures, length of the culture period, temperature, light quality, and light intensity can eventually result in several developmental and physiological aberrations of the micropropagated plants [ 60 ]. The term “somaclonal variation” refers to any phenotypic variation detected among micropropagated plants [ 151 ].…”

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

“…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.…”

“…In C. sativa, the majority of micropropagation studies rely on shoot multiplication from existing meristems found in the apical and axillary buds [6]. Numerous protocols report regeneration from meristematic tissues at levels greater than 70% [4,[7][8][9], with the most effective of these protocols rely on existing meristems found in the nodal tissues of vegetative cuttings.…”

Plant tissue culture of Nicotiana tabacum cv. TAPM 26 and its minimum inhibition against herbicide-Dalapon

“…BAP was essential factor for shoot proliferation, whereas poor shoot development occurred on the media without cytokinins. In fact, certain explants will be more responsive than the others for instance the cotyledon of some species regenerate shoot better than true leaves or stems [39,52].…”