A non-destructive plant screening method for improving sample uniformity in horticultural crops based on a hydrogen peroxide fluorescent probe

Introduction Hydrogen peroxide (H 2 O 2 ) functions as a key signaling molecule in plants responding to stress. Although numerous detection methods have been developed, simple and non-destructive techniques for the semi-quantitative monitoring of H 2 O 2 in plant tissues remain scarce. Methods In this study, we developed a "turn-on" fluorescent probe specifically designed to detect endogenous H 2 O 2 in plant tissues, and conducted spectroscopic and in vivo toxicity tests. Furthermore, under experimentally controlled stress conditions, we utilized this probe to detect H 2 O 2 levels in four distinct plant types exposed to salt, waterlogging, cadmium, and drought stresses. Additionally, H 2 O 2 was detected in a grafting model under non-experimentally controlled stress conditions. Results The results showed that the probe demonstrated excellent selectivity, a strong linear correlation (R2 = 0.9849), and a low detection limit of 0.6450 μmol/L. Importantly, it exhibits good biocompatibility with plant tissues and effectively minimizes detection errors caused by transient H 2 O 2 fluctuations induced by environmental changes. Consequently, it provides more accurate and stress-reflective H 2 O 2 measurements. Under experimentally controlled stress conditions, the changes in relative fluorescence intensity conformed to the typical response patterns observed when plants experience graded levels of stress. Notably, even under complex grafting conditions without imposed stress gradients, applying the probe to bottle gourd (Lagenaria siceraria) rootstocks with different graft compatibility produced fluorescence dynamics consistent with the typical H 2 O 2 responses of compatible and incompatible rootstocks, and the distribution of relative fluorescence intensity within the population underscored the importance of prescreening plants for biological studies. Pearson correlation and Bland-Altman analyses confirmed good agreement between our method and the commercial assay kit. Discussion These results demonstrate that the LWS probe enables H 2 O 2 detection and, in combination with the IVIS in vivo imaging system, can screen individual plants differing in stress responses more effectively than other sensors. This non-destructive approach preserves the structural integrity of plant samples, enabling follow-up physiological, biochemical, and genomic analyses on the same specimens. This method provides a reliable prescreening platform for investigating plant stress responses at the biological level.