High-Throughput Hypocotyl Grafting and Vascular Reconnection Assessment in Arabidopsis Seedlings

Plant grafting is a powerful tool in research and agriculture, enabling the study of long-distance signaling and improving crop performance. Hypocotyl grafting in Arabidopsis thaliana has become a key method for investigating inter-tissue communication, root-to-shoot transport, and vascular regeneration. Here, we describe a high-throughput hypocotyl grafting protocol that consistently achieves success rates above 90% while minimizing complications, such as adventitious root formation, through an optimized hypocotyl cutting position. Our streamlined approach eliminates the need for silicone tubes and fine forceps, requiring only a standard scalpel for the cutting and joining procedure. With proper training, an experienced user can graft up to 1000 plants per day, facilitating large-scale experiments. Additionally, we present vascular connectivity assays using fluorescent dyes and the adjuvant Adigor to enable phloem and xylem transport analysis across the graft junction without the need for wounding. By simplifying the grafting process while maintaining high efficiency, our protocol makes Arabidopsis grafting more accessible and scalable, facilitating research on long-distance molecular transport, vascular development, and plant biotechnology applications.