Mix-and-match: an improved, fast and accessible protocol for hypocotyl micrografting of Arabidopsis seedlings with systemic ACC responses as a case study

BACKGROUND: Grafting is a technique widely used in horticulture that also has been applied in agriculture. In plant physiology, grafting facilitates the elucidation of mechanisms underlying growth and developmental processes, through the construction of chimeric plants with organs of different genotypes. Despite its small size, the model species Arabidopsis thaliana is very amenable for grafting, which can be useful to investigate transport of nutrients, amino acids or secondary metabolites between different tissues, or to investigate developmental processes depending on root-to-shoot communication, such as shoot branching, root and shoot plasticity upon shade avoidance, or disease resistance. Nevertheless, grafting protocols are usually technically challenging and training is required to achieve a reasonable success rate. Additionally, specialized tools and equipment are often needed, such as chips to accommodate the grafted plantlets or collars to maintain the contact between root and shoot. RESULTS: In this methodology paper, we provide a fast, easy, accessible, and specialized equipment-free protocol that enables high success ratios. Critical steps and notes are detailed, easing the implementation of the procedure for non-trained researchers. An example of the protocol application by three independent non-trained researchers shows that this method allows to achieve a 90–100% of grafting efficiency after 6 days post-grafting recovery. In addition, the grafting of Col-0 with the acs8x mutant, depleted in 1-aminocyclopropane-1-carboxylic acid (ACC), the biosynthetic precursor of the phytohormone ethylene, provides an example of the application of this optimized protocol, showing the suitability of the process to study long-distance transport processes. CONCLUSIONS: We present an optimized protocol for hypocotyl grafting of 4-day-old Arabidopsis thaliana seedlings. The combination of conditions yields a grafting success of 90–100% and provides an easy and accessible methodology, reducing the time frame, and without the necessity of acquiring specialized equipment. The presented protocol is simple, fast and highly efficient, easing the inclusion of hypocotyl grafting assays in any research project. In addition, the description of the protocol is detailed to a level ensuring that even non-trained researchers, are sufficiently prepared to adopt the grafting methodology.