Practical Guide to In Vitro Clonal Propagation of <em>Nicotiana benthamiana</em> Using Axillary Shoot Induction

This protocol outlines a reliable method for the micropropagation of <i>Nicotiana benthamiana</i> using axillary shoot branching. Axillary shoot induction involves stimulating the outgrowth of dormant buds located at the leaf axils, allowing for the development of genetically stable shoots without callus formation or the use of exogenous plant growth regulators. Nodal explants are cultured on MS medium supplemented with kinetin and indole-3-butyric acid (IBA) to induce shoot formation. Isolated shoots are then transferred to hormone-free MS medium for rooting. This method is simple, reproducible, and supports rapid plant multiplication for downstream applications such as agroinfiltration or transient protein expression. Key features • Axillary branching-based micropropagation enables efficient regeneration of <i>N. benthamiana</i> from nodal explants, supporting a cyclic system for continuous in vitro plant supply [1]. • Rooting occurs on hormone-free medium, with even shoots as small as 0.5 mm readily forming roots, simplifying and accelerating the propagation process. • Root formation begins within 7 days, including in transgenic lines that are typically difficult to root using conventional methods. • Fully developed, rooted plants are ready within 4 weeks, ideal for transient expression assays, recombinant protein production, and secondary metabolite extraction.