Protocols for Root Developmental Studies in White Lupin.

White lupin (Lupinus albus) is a legume species that develops two remarkable root adaptations to nutrient limitation: nitrogen-fixing nodules and dense cluster roots specialized for phosphate acquisition. These traits, together with the species' agronomic relevance and strong developmental plasticity, make white lupin a powerful model for studying local and systemic regulation of root development under nutrient stress. This chapter provides a comprehensive collection of experimental procedures designed to investigate cluster root formation, nodulation, and associated physiological responses at both cellular and whole-plant levels. We describe optimized hydroponic culture systems that allow precise control of nitrogen and phosphate availability, together with reliable seed sterilization and germination procedures. We present a robust protocol for Agrobacterium rhizogenes-mediated hairy-root transformation to investigate gene function in root tissues. A controlled nodulation assay using Bradyrhizobium lupini enables reproducible symbiotic infection and nodule development. A grafting method is also provided to dissect local versus systemic contributions to root architectural responses. Finally, we detail three in situ physiological assays-phosphatase activity, ferric reductase activity, and proton efflux-that collectively allow the visualization of key functional traits associated with nutrient foraging. Together, these protocols provide an integrated toolkit for exploring the developmental and physiological mechanisms underpinning root plasticity and symbiotic regulation in white lupin.