Beyond Averages: Chitosan Dispersity Affects the Bioactivity Reproducibility on In Vitro Maize (var. Jubilee)...

Chitosan bioactivity has been widely evaluated in seed germination; however, its effects remain inconsistent. Some studies report biostimulant effects, while others manifest inhibitory effects, and some show no effect on germination. A major factor contributing to this poor reproducibility appears to be the variation in the degree of polymerization (X), the molar fraction of acetylation (f_A), and the chitosan concentration. However, it remains unclear whether controlling these parameters alone is sufficient to ensure consistent bioactivity in conventional polymeric chitosan samples. To elucidate this, maize seeds (Zea mays L. var. Jubilee) were soaked in chitosan solutions (pH 5) at concentrations ranging from 0.25 to 1.00% w/w for 4 h, as per the literature, to evaluate their effect on germination compared to a control (water). Nine chitosan samples were tested, differing in X (5558 to 17,161) and f_A (0.07 to 0.33). After germination, several response factors were measured, including fresh and dry biomass, root number, and root and epicotyl length. The results showed that chitosans with higher X values (9134 to 17,161) inhibit germination, decreasing it by up to a value of 3% ± 6 at 1.00% w/w. Chitosans with a lower X (5694 ± 131) exhibited a reduced inhibitory effect (63% ± 6 to 100%) across all evaluated concentrations. None of the treatments showed biostimulation; instead, chitosan consistently delayed the germination rate compared to the control, confirming an inhibitory effect regardless of the final percentage. Nevertheless, while X and concentration establish the general inhibitory trends, considerable residual variability suggests that these factors alone are insufficient to ensure consistent bioactivity. A Gauge Repeatability and Reproducibility (R&R) analysis provides preliminary evidence suggesting that molecular heterogeneity, specifically dispersity (Ð_X), is a key factor driving the observed inconsistencies and significantly affecting the reproducibility of the results within the scope of this study.