Development of a robust transient expression screening system in protoplasts of Cannabis

L Matchett-Oates,Ehab Mohamaden,G C Spangenberg,N O I Cogan

doi:10.1007/s11627-021-10178-0

Abstract

Transient expression systems in mesophyll protoplasts have been utilised in many plant species as an indispensable tool for gene function analysis and efficacious genome editing constructs. However, such a system has not been developed in Cannabis due to the recalcitrant nature of the plant to tissue culture as well as its illegal status for many years. In this study, young expanding leaves from aseptic in vitro Cannabis explants were used for protoplast isolation. Factorial designs were used to optimise variables in viable protoplast isolation and transient expression of GFP, with a range analyses performed to determine, and quantify, significantly impacting variables. Viable protoplast yields as high as 5.7 × 106 were achieved with 2.5% (w/v) Cellulase R-10, 0.3% (w/v) Macerozyme R-10 and 0.7 M mannitol, incubated for 16 h. As indicated by the transient expression of GFP, efficiency reached 23.2% with 30 μg plasmid, 50% PEG, 1 × 106 protoplasts and a transfection duration of 20 min. Application of the optimised protocol for protoplast isolation was successfully evaluated on three subsequent unrelated genotypes to highlight the robustness and broad applicability of the developed technique.

Highlights

Cannabis sativa L. (Cannabis) is a highly polymorphic, wind pollinated herb originating in China where evidence of its cultivation dates back to 4000 BC (Zuardi 2006)
Mesophyll Protoplast Isolation From Cannabis Leaf Tissue Initially, protoplasts were generated from rapidly expanding leaves from 1- to 2-mo-old in vitro plantlets that were used as the source material (Fig. 1a, b)
Plantlets cultured at 26°C in 1⁄2 MS medium with 5 μM IBA were used for mesophyll protoplast isolation

Summary

Introduction

Cannabis sativa L. (Cannabis) is a highly polymorphic, wind pollinated herb originating in China where evidence of its cultivation dates back to 4000 BC (Zuardi 2006). (Cannabis) is a highly polymorphic, wind pollinated herb originating in China where evidence of its cultivation dates back to 4000 BC (Zuardi 2006). Chromosomal resolution of the Cannabis genome was published along with other genetic resources for identification of important cannabinoid biosynthesis genes (Grassa et al 2018; Laverty et al 2019). With the recent release of the chromosomal assembly and high-density linkage map of the Cannabis genome (Grassa et al 2018; Laverty et al 2019), identification of genes involved in cannabinoid biosynthesis is relatively straightforward, with benchmark standards being developed for genetic engineering of these genes (Matchett-Oates et al 2020.). To date, no procedures for the evaluation of genes

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Conclusion