Inflorescence stem grafting made easy in Arabidopsis

Abstract

UNLABELLED BACKGROUND Plant grafting techniques have deepened our understanding of the signals facilitating communication between the root and shoot, as well as between shoot and reproductive organs. Transmissible signalling molecules can include hormones, peptides, proteins and metabolites: some of which travel long distances to communicate stress, nutrient status, disease and developmental events. While hypocotyl micrografting techniques have been successfully established for Arabidopsis to explore root to shoot communications, inflorescence grafting in Arabidopsis has not been exploited to the same extent. Two different strategies (horizontal and wedge-style inflorescence grafting) have been developed to explore long distance signalling between the shoot and reproductive organs. We developed a robust wedge-cleft grafting method, with success rates greater than 87%, by developing better tissue contact between the stems from the inflorescence scion and rootstock. We describe how to perform a successful inflorescence stem graft that allows for reproducible translocation experiments into the physiological, developmental and molecular aspects of long distance signalling events that promote reproduction. RESULTS Wedge grafts of the Arabidopsis inflorescence stem were supported with silicone tubing and further sealed with parafilm to maintain the vascular flow of nutrients to the shoot and reproductive tissues. Nearly all (87%) grafted plants formed a strong union between the scion and rootstock. The success of grafting was scored using an inflorescence growth assay based upon the growth of primary stem. Repeated pruning produced new cauline tissues, healthy flowers and reproductive siliques, which indicates a healthy flow of nutrients from the rootstock. Removal of the silicone tubing showed a tightly fused wedge graft junction with callus proliferation. Histological staining of sections through the graft junction demonstrated the differentiation of newly formed vascular connections, parenchyma tissue and lignin accumulation, supporting the presumed success of the graft union between two sections of the primary inflorescence stem. CONCLUSIONS We describe a simple and reliable method for grafting sections of an Arabidopsis inflorescence stem. This step-by-step protocol facilitates laboratories without grafting experience to further explore the molecular and chemical signalling which coordinates communications between the shoot and reproductive tissues. Show more