Quantifying Arbuscular Mycorrhizal Fungal Colonization via Anthocyanin Pigmentation in Medicago truncatula Roots

Plant responses to environmental stimuli are often shaped by a history of previous interactions, forming the foundation for stress memory and adaptive plasticity. Arbuscular mycorrhizal (AM) fungi establish a mutualistic relationship with most land plants, enhancing nutrient uptake and stress resilience, and are increasingly recognized as biological agents contributing to plant stress memory. However, quantifying AM colonization, especially in large-scale or time-course experiments investigating priming or memory effects, remains a technical bottleneck. Conventional staining methods are time-consuming, destructive, and incompatible with live imaging. This chapter presents a robust, nondestructive, and quantitative protocol to assess AM colonization in Medicago truncatula roots using a visible anthocyanin pigmentation marker. The method employs a synthetic construct expressing the R2R3 MYB transcription factor MtLAP1, driven by the AM-inducible Kunitz Protease Inhibitor 106 (KPI106) promoter, enabling visualization of arbuscule-containing root cells through purple/red pigmentation. The protocol encompasses Agrobacterium rhizogenes-mediated hairy root transformation, standardized mycorrhization assays, and anthocyanin pigment extraction and quantification. Anthocyanin accumulation correlates strongly with conventional staining-based colonization estimates, and the system enables early detection, live imaging, and high-throughput screening of mutants with altered AM phenotypes. This method offers a powerful tool for dissecting the functional role of mycorrhizal symbiosis in plant stress memory and is especially suited for forward genetic screens, stress priming experiments, and live-tracking of root-fungus interactions over time.