A Retention-Based Phenotyping Protocol for Identifying Soybean Accessions with Superior BNF-Associated Performance

Biological nitrogen fixation (BNF) is an important trait in soybean (Glycine max L. Merr.) because it reduces dependence on synthetic N fertilizers and supports sustainable crop production; however, field-based phenotyping methods for evaluating BNF-associated performance are often laborious, costly, and difficult to apply to large germplasm populations. Here, we developed a retention-based phenotyping protocol using 194 soybean accessions from maturity groups III–VII evaluated under non-N-applied (N−) and N-applied (N+) field conditions during two growing seasons. Canopy chlorophyll content was monitored using SPAD measurements, whereas 1000-seed weight and seed protein concentration were determined at harvest. Trait performance under N− conditions was normalized relative to N+ conditions using retention values. Mean SPAD retention, 1000-seed weight retention, and protein concentration retention were integrated into a Composite Retention Index (CRI) to classify accessions across contrasting N environments. Indeed, CRI effectively separated accessions into three tiers (high, intermediate, and low retention) and captured variation in canopy chlorophyll retention, 1000-seed-weight retention, and protein concentration retention. Overall, the proposed protocol provides a practical, economical, and reproducible field-based approach for identifying soybean accessions with superior BNF-associated performance and may serve as a useful phenotyping tool in genetic improvement programs.