Optimizing sap sampling methodologies for nutrient diagnostics in lettuce and tomato grown under controlled environment agriculture

Effective nutrient management is crucial for maintaining growth and yield in controlled environment agriculture (CEA). However, nutrient interpretation depends on sampling methodology, and standardized sampling protocols for sap analysis remain inconsistent. This study evaluated five key sap-sampling methods: leaf number, tissue age, tissue part (only for tomatoes, Solanum lycopersicum L.), collection time, and sampling frequency, to establish sampling protocols for hydroponic lettuce ( Lactuca sativa ) and tomato in CEA. A split-plot design with repeated measures was employed using three cultivars per crop, and nutrient concentrations were analyzed within each cultivar using linear mixed-effects models with treatment, stage, and their interaction (treatment × stage), with rep included as a random effect. Two sap-extraction methods were employed: mechanical tissue crushing (TC) and potassium chloride (KCl) extraction. Nutrient leaf analysis (NLA) was subsequently used as the comparative reference method representing total tissue nutrient status. In lettuce, sampling effects were generally modest and nutrient- and cultivar-specific, and responses were frequently not conserved between TC and KCl. Effects of leaf number were more evident under KCl extraction than under TC. Tissue-age effects were present but expressed differently by extraction method; time-of-collection effects were small and inconsistent, and stage effects over the short cycle, 12 vs. 24 days after transplantation (DAT), were limited and method-dependent. In tomatoes, sampling effects were stronger and more structured. The number of leaves interacted with the developmental stage, and sap inorganic N consistently showed a leaf number × stage interaction across cultivars under both TC and KCl, indicating that leaf number must remain constant across DAT for valid interpretation. Tissue age produced strong separation with cultivar-specific age × stage interaction patterns across methods, and in tissue part, we found part × stage interactions, confirming that blade and petiole sap are not interchangeable. Sampling frequency affected tomato sap nutrient profiles across 63–253 DAT, with TC capturing short-term nutrient pools and KCl more clearly reflecting long-term enrichment patterns.