Medical Cannabis and Industrial Hemp Tissue Culture: Present Status and Future Potential

Abstract

In recent years high-THC (psychoactive) and low-THC (industrial hemp) type cannabis (Cannabis sativa L.) have gained immense attention in medical, food, and a plethora of other consumer product markets. Among the planting materials used for cultivation, tissue culture clones provide various advantages such as economies of scale, production of disease-free and true-to-type plants for reducing the risk of GMP-EuGMP level medical cannabis production, as well as the development and application of various technologies for genetic improvement. Various tissue culture methods have the potential application with cannabis for research, breeding, and novel trait development, as well as commercial mass propagation. Although tissue culture techniques for plant regeneration and micropropagation have been reported for different cannabis genotypes and explant sources, there are significant variations in the response of cultures and the morphogenic pathway. Methods for many high-yielding elite strains are still rudimentary, and protocols are not established. With a recent focus on sequencing and genomics in cannabis, genetic transformation systems are applied to medical cannabis and hemp for functional gene annotation via traditional and transient transformation methods to create novel phenotypes by gene expression modulation and to validate gene function. This review presents the current status of research focusing on different aspects of tissue culture, including micropropagation, transformation, and the regeneration of medicinal cannabis and industrial hemp transformants. Potential future tissue culture research strategies helping elite cannabis breeding and propagation are also presented.

Keywords: Cannabis sativa; hemp; in vitro; micropropagation; tissue culture.

FIGURE 1

Cannabis leaf showing morphological differences of the three different species (C. indica, C. sativa, and C. ruderalis).

FIGURE 2

Hemp nodal cloning. (A) Hemp plants at 6–8 leaf stage. (B) Elongated lateral branches after terminal buds removed from female plants (C) lateral branches planted in soil after excision from mother plants and. (D) Vegetative clones transferred to 7-inch pots after roots were established and grown. (E) Vegetative clone at maturity.

FIGURE 3

Hemp tissue culture propagation. (A) Hypocotyl explants on callus-induction media. (B) Hypocotyl explants with the callus on callus induction media. (C,D) Callus and developing shoots on shoot-induction media. (E) Developed shoots on root-induction media.

FIGURE 4

Evolution of cannabis tissue culture research. The green curved arrow on the left shows the key events in cannabis use. Each rectangle on the right shows the major research and development activities at different years. Each brown arrow indicates that the technology is continuously developing and research work is in progress in the particular research area.

FIGURE 5

Developmental pathways observed in C. sativa (industrial hemp) microspore culture. (A–C) Male gametophyte development in C. sativa during in vitro culture. (A) Uninucleate microspores; (B) uninucleate microspores after 3 days in culture media; (C) symmetrically divided microspore with two equally sized nuclei; (D) multinucleate structure without organization and still enclosed in exine; (E) globular multicellular structure with developing exine; and (F) heart-shape embryo with two distinct domains. The nuclei in (A–C) are stained with the nuclear dye 4′,6-diamidino-2-phenylindole (DAPI) to indicate viability.

FIGURE 6

General schematic diagram showing steps for calcium chloride encapsulated synthetic seed production.

FIGURE 7

A flow chart depicting different approaches that can be used to determine the genetic stability of in vitro regenerated or conserved cannabis plants, compared to its donor counterparts.

FIGURE 8

Integration of automation and bioreactor technologies for mass propagation in cannabis for low cost clonal multiplication at in vitro level.