Optimizing in vitro germination of Amaranthus hypochondriacus via a resolution V 2^5-1 fractional factorial design

Amaranth seed germination is highly sensitive to early handling and incubation conditions, yet the relative contribution of key pre-germination and growth variables is rarely quantified within a single screening framework. In this study, a resolution V fractional factorial design 2^5-1 with four center points (20 runs, no replicates) was used to identify the main factors that influence germination rate (%) and root length after five days of germination. Five factors were evaluated: medium (Murashige and Skoog, MS, with or without 1% sucrose), number of seeds per square dish (20, 25 as central point, 30), photoperiod (short 8 h light/16 h dark vs long 16 h light/8 h dark), hypochlorite washing time (3.5% v/v; 5, 10, 15 min), and cold stratification, (1, 2, 3) days at 4°C. Germination rate was strongly affected by photoperiod and medium, with washing time also significant; several second-order interactions were detected, indicating non-additive behavior within the explored region. For root length, a Box–Cox analysis supported a square-root transformation, and only medium and photoperiod remained significant. Multi-response desirability optimization identified MS (no sucrose), 20 seeds, a short photoperiod, 5 minutes of washing, and 1 day of cold stratification as the best conditions. Confirmation runs validated germination predictions (87.0 ± 3.6% vs 87.39% predicted), while root length was overpredicted (-18.3% relative error), consistent with higher biological variability and the screening nature of the design. Overall, photoperiod emerged as the dominant factor controlling both responses, highlighting light regime as a primary variable for improving early amaranth seeds establishment under in vitro conditions.