Working Paper Article Version 1 This version is not peer-reviewed

Cryopreservation of 13 Commercial Cannabis Sativa Genotypes Using in Vitro Nodal Explants

Version 1 : Received: 17 July 2021 / Approved: 19 July 2021 / Online: 19 July 2021 (18:13:48 CEST)

How to cite: Downey, C.D.; Jones, A.M.P. Cryopreservation of 13 Commercial Cannabis Sativa Genotypes Using in Vitro Nodal Explants. Preprints 2021, 2021070426 Downey, C.D.; Jones, A.M.P. Cryopreservation of 13 Commercial Cannabis Sativa Genotypes Using in Vitro Nodal Explants. Preprints 2021, 2021070426

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 hours in a pre-culture solution (PCS), followed by a 20 minute treatment in a loading solution (LS), and a 60 minute 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.

Subject Areas

Cannabis sativa; Germplasm preservation; Droplet vitrification; Conventional vitrification; Tissue culture

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