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.

Keywords: Cannabis; DKW; TDZ; floral reversion; marihuana; marijuana; micropropagation; regeneration; review; tissue culture.

Figure 1

In Vitro flowering of Cannabis sativa. (A) Flowering C. sativa male plant displaying a hermaphroditic phenotype, showing female flowers (left) adjacent to male flowers (right). Scale bar—1 mm. (B) In Vitro male inflorescences of C. sativa. Scale bar—1 mm (C) A pair of female C. sativa florets obtained from In Vitro flowering C. sativa. Scale bar—1 mm. (D) Glandular trichomes developing on the bract surrounding the ovary of a female C. sativa inflorescence. Scale bar—2 mm. (E) Mature flowering In Vitro explant of C. sativa. Scale bar—1 cm. (F) Four-week-old vegetative explants reverted from In Vitro C. sativa inflorescences. Scale bar—1 cm.

Figure 2

(A) In Vitro germinated seedling of C. sativa demonstrating opposite leaf arrangement. White arrows show oppositely oriented first true leaves. Scale bar—1 cm. (B) A Stage 2 vegetative explant (subcultured from a nodal explant) of C. sativa demonstrating alternate leaf arrangement (black arrows), a change in phyllotaxy resulting from explant maturation. Scale bar—1 cm. (C) C. sativa grown in controlled environment growth chambers under fluorescent lighting. (D) C. sativa grown outdoors under a shade cloth in Colombia. Image supplied courtesy of Avicanna™. (E) Hyperhydric C. sativa explants growing on Murashige and Skoog (MS) medium supplemented with 0.5 µM thidiazuron (TDZ).

Figure 3

Healthy C. sativa explants growing in We-V boxes (A). Callus cultures growing in glass culture vessels under LED lighting in a controlled environment growth chamber (B) and high-density stackable culture vessels (We-V) with individually programmable LED lighting (C) demonstrate the variety and density with which C. sativa can be cultured under In Vitro conditions.

Figure 4

The five-stage micropropagation process for tissue culture. The red arrow is indicative of the 1-3-4 approach (where Stage 2 is skipped). Stage 2 is commonly skipped in C. sativa micropropagation methods due to the plant’s recalcitrance to long-term culture characterized by a slow decline in fitness. Inclusion of Stage 2 allows for repeated subcultures of In Vitro plants (indicated by the circular arrows), therefore facilitating large-scale multiplication or long-term germplasm storage.

Figure 5

Tissue culture of healthy explants relies on the careful optimization of multiple factors. Center image: Freshly subcultured vegetative explant of C. sativa.

Figure 6

Yearly publications and the number of citations for articles matching the search TOPIC: (Cannabis sativa OR hemp) AND TOPIC: (regeneration) AND TOPIC: (micropropagation OR In Vitro OR tissue culture) on the Web of Science database. Data obtained from Web of Science® on 17 November 2020 using Microsoft Edge®. Data presented may not be a comprehensive representation of the available publications and are limited to publications indexed by Web of Science®.