Introducing Transformative Plant Biotechnology: Establishing a transient genetic transformation protocol in Cannabis sativa

Abstract

*Cannabis sativa* is a multipurpose crop with numerous beneficial applications for people and the planet. The uses of Cannabis range from the pharmaceutical sector to the building materials industry. Agrobacterium-mediated transient transformation is a highly useful method for gene function studies, and has been established in many diverse plant species. However, because of the widespread ban of Cannabis cultivation and research over the past century, a high-throughput and reproducible transient transformation system in Cannabis has not been established and optimised to the same extent as for other plant species. Here, we describe a protocol for Agrobacterium tumefaciens mediated transient transformation in Cannabis leaves, using the RUBY reporter construct, which encodes the enzymes in the betalain biosynthesis pathway. The protocol utilises an “agroinjection” method, whereby Agrobacterium is injected into Cannabis leaves. To optimise this protocol, different induction buffers, cultivars of Cannabis, and developmental stages of Cannabis plants were tested to establish the optimal conditions to achieve reliable transient transformation. Transformation efficiency was determined by quantifying the amount of betalain pigment present in the transformed leaf tissue. Further, this protocol was tested for its efficacy for protein subcellular localisation assays using fluorescent protein markers. This method is an efficient and easy way of determining transformation efficiency and selecting transformants in Cannabis without specialised equipment, and can be used for assessing gene function and protein localisation assays. **Co-Authors**: Seán Kettle, Aditya Nayak, Alice Destailleur, Rainer Melzer, Susanne Schilling