In Exp. 1 and Exp. 2, the phenotypic data measured on leaf physiological traits (Chlorophyll content, stomatal content, osmotic potential, and leaf water potential), shoot and root fresh, and dry biomass were analyzed using the GLM approach with “blocks” as a fixed variable. Step-wise regression and correlation analyses between traits measured were also performed using SAS version 9.1. The image-based analysis of alfalfa three leaflet blade (area, length, width, entropy, and compactness) and root (diameter, volume, width, angle, and vertical root length) traits were performed using methods described earlier (Liao et al., 2017 (link); Paez-Garcia et al., 2019 (link)). Analysis of variance (ANOVA) of shoot (NDVI) and root traits (median, maximum number of roots, total root length, max width, network area, solidity, perimeter, average radius, volume, surface area, maximum radius, steep angle frequency, and holes) from raised-bed experiment was analyzed using ProcGLM analysis in SAS version 9.1, and their relationships between traits were performed using SAS JMP version 15 program. In all the experiments, the least significant difference (LSD) at α = 0.05 was used to determine significant differences among genotypes, treatments, and the interaction. Statistical significance was based on a p-value of 0.05.
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