Abstract

Cannabis has developed into a multi-billion-dollar industry that relies on clonal propagation of elite genetics with desirable agronomic and chemical phenotypes. While the goal of clonal propagation is to produce genetically uniform plants, somatic mutations can accumulate during growth and compromise long-term genetic fidelity. Cryopreservation is a process in which tissues are stored at cryogenic temperatures, halting cell division and metabolic processes to facilitate high fidelity germplasm preservation. In this study, a series of experiments were conducted to optimize various stages of cryopreservation and develop a protocol for long-term germplasm storage of Cannabis sativa. The resulting protocol uses a standard vitrification procedure to cryopreserve nodal explants from in vitro shoots as follows: nodes were cultured for 17 h in a pre-culture solution (PCS), followed by a 20-min treatment in a loading solution (LS), and a 60 min incubation in plant vitrification solution 2 (PVS2). The nodes were then flash frozen in liquid nitrogen, re-warmed in an unloading solution at 40 °C, and cultured on basal MS culture medium in the dark for 5 days followed by transfer to standard culture conditions. This protocol was tested across 13 genotypes to assess the genotypic variability. The protocol was successful across all 13 genotypes, but significant variation was observed in tissue survival (43.3–80%) and regrowth of shoots (26.7–66.7%). Plants grown from cryopreserved samples were morphologically and chemically similar to control plants for most major traits, but some differences were observed in the minor cannabinoid and terpene profiles. While further improvements are likely possible, this study provides a functional cryopreservation system that works across multiple commercial genotypes for long-term germplasm preservation.

Highlights

  • A major challenge facing the development and application of plant based medicines, including cannabis, is producing material with a consistent and well described chemical profile to ensure predictable and reproducible biological effects [1]

  • shoot multiplication media (SMM) media— generally used for maintenance of in vitro donor plants—was supplemented with KIN and naphthaleneacetic acid (NAA), which have been documented in inducing shoot proliferation and root induction in micropropagated samples, respectfully [26]

  • The results presented in this study showed no significant differences in sample survival or recovery in respect to the vitrification method used, and recovery media was only significant for sample recovery when the droplet vitrification method was used

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Summary

Introduction

A major challenge facing the development and application of plant based medicines, including cannabis, is producing material with a consistent and well described chemical profile to ensure predictable and reproducible biological effects [1]. Cannabis produces a wide range of other compounds such as terpenes, which are responsible for the flavor and aroma profile of the product and are thought to interact with cannabinoids to modify their biological activity [3,4]. This complexity introduces a significant challenge in producing uniform products, which is required to ensure consistent medicinal activity and product quality. While the challenge of chemical standardization is not unique to cannabis, most governments have imposed strict quality assurance regulations that require careful attention to product quality and uniformity

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