Feasibility Study of Using Ca2+ Flow Rate to Characterize Eelgrass (Zostera marina) Seed Viability

Abstract:

Eelgrass (Zostera marina), a perennial marine seed plant of Magnoliaceae, is commonly found in offshore shallows and river inlets and lives in submerged water. Eelgrass has important ecological services, such as water purification, protection of biodiversity, dike protection, disaster mitigation, and carbon sequestration. In recent years, with the increasing intensity of marine development and utilization and the impact of global climate change, seagrass bed resources have shown signs of increasing decline. The degradation rate of, China's seagrass beds also accelerates annually. Thus, the protection and restoration of seagrass bed resources cannot be delayed. In addition to taking effective management measures, scientific restoration of seagrass beds through human intervention is another important approach to protect existing seagrass beds. Transplanting artificially cultivated seagrass seedlings for seagrass bed restoration is also a way to utilize the seeds efficiently, and the evaluation of seed vigor status is the key to determine the germination rate and seedling establishment rate. Seed vigor is an important index for screening high germination rate, high seedling emergence rate, and other high-quality varieties. It is also the main index reflecting the rapid and neat emergence of seeds and the normal growth of seedlings. At present, methods commonly used to test eelgrass seed vigor are low-temperature germination assay, conductivity assay, enzyme vigor assay, and 2,3,5-tripheyl tetrazolium chloride (TTC) staining assay. However, low-temperature germination test cannot reflect the real vigor level of seeds well, especially in eelgrass seeds, because the germination time needs more than 2 weeks. Seedling growth determination, germination rate determination, and other traditional methods for detecting seed vigor need to be verified by a large number of repetitive experiments, which require large amounts of manpower, material resources, and time, as well as a large amount of investment in the development and development of eelgrass seeds. Similarly, conductivity measurement, enzyme activity measurement, and seedling growth rate measurement need to be validated by a large number of repetitive tests, which require a large investment of labor, material, and time, and may also damage seed samples. With the rapid development of technology, various non-contact, non-destructive, rapid seed viability testing methods have emerged. These methods include non-invasive micro-measurement, near-infrared spectroscopy, hyperspectral imaging, electronic nose detection. Among them, non-invasive micro-measurement determines seed viability by means of the sample. It also determines seed vigor by measuring the ion or molecular flow rate of drops on the seed surface. Given its advantages of non-damage, multi-electrode, multi-angle, high sensitivity, and high resolution, this technique has been applied in different plant research fields, such as plant salt resistance, plant pathology, and plant heavy metal resistance. In this study, we determined the Ca²⁺ flow rate and direction in eelgrass seeds with different activities obtained from drying treatment by a non-invasive microbolometer system to investigate the relationship between Ca²⁺ flow rate and eelgrass seed vigor, and provide a new method for the rapid, non-invasive, and in vivo identification of eelgrass seed vigor. Prior knowledge of seed viability status is a crucial aspect of artificial seedling cultivation, including eelgrass. In this study, eelgrass seeds were subjected to different degrees of drought stress for their special recalcitrant properties, and the same batch of eelgrass seeds was artificially treated to create differences in vigor. While different indicators were used to describe the physiological state of the seeds after the drying treatment, non-invasive micrometry was used to determine the Ca²⁺ flow rate of the seeds and investigate the relationship between eelgrass seed vigor and Ca²⁺ flow rate. In this study, drying treatments were used to artificially create viability differences in eelgrass seeds from the same batch, totaling five drying times (0, 1, 2, 4, and 8 h) and 20 groups of samples. Germination rate, relative conductivity, water content, catalase activity, and malondialdehyde content were determined. Non-invasive micro-measurement was applied to the detection research of eelgrass seed vigor. Its primary objectives were to verify the feasibility of seed vigor grading through preliminary experiments, formulate a demonstration scheme, and further lay a solid foundation for the subsequent establishment of a standardized system for eelgrass seed vigor grading. Results showed that the germination rate gradually decreased and the relative conductivity increased with treatment time, the germination rate of the seeds after 4 h of treatment was 12% lower than that of the untreated seeds, and the germination rate after 8 h of treatment significantly reduced and was 68.7% lower than that of the untreated seeds. Catalase activity also significantly changed with treatment time. The Ca²⁺ was effluxed, and the efflux rate increased with treatment time. The germination rate and Ca²⁺ efflux flow rate were significantly negatively correlated, and the fitted linear equation was y = –0.192 2x + 94.09, with an R² of 0.860 6. This study proved that the Ca²⁺ flow rate could serve as an eelgrass seed vigor detection index, providing a basis for the rapid and non-destructive identification of eelgrass seed vigor.