Nodule color and morphology are key readouts of legume symbiotic performance. However, long-term preservation of post-excavation nodules with intact morphology, color, and microbial cleanliness remains a major challenge. This study developed a two-stage aqueous-phase preservation method (TAPP) that enables rapid structural fixation and long-term chemical stabilization. A comprehensive evaluation was subsequently established, incorporating composite morphological score (0–5), color difference (ΔE) and its piecewise slope over time (\(\:{\gamma\:}_{\varDelta\:E}\)), and visible contamination grade (0–3). Peanut and soybean nodules from multiple regions and cultivars were tracked for 24 months under five preservation methods: TAPP, FormalinCu, TAPP-Resin, Resin, and AirDry. TAPP showed the best overall preservation, with composite morphological scores of 4.65 ± 0.14 for peanut and 4.63 ± 0.22 for soybean at 24 months, and no visible mold. Color change slowed over time: \(\:{\gamma\:}_{\varDelta\:E}\) decreased from 1.83 to 1.10 ΔE·month− 1 during 0–1 month to 0.16 ΔE·month− 1 during 12–24 months, yielding final ΔE values of 10.53 ± 1.88 and 10.32 ± 1.93, respectively. Notably, TAPP pretreatment markedly improved resin-embedded samples, demonstrating scalability and flexible deployment. In addition, this study further proposes a stage-wise workflow that integrates on-site pre-fixation, long-distance transport, and long-term storage to enable cross-regional circulation and collaborative phenomics of oxidation-prone, dehydration-sensitive nodules. Together, this work establishes a standardized, traceable workflow to preserve and benchmark legume root nodule phenotypes, supporting cross-laboratory comparability and longitudinal cross-source analyses.
