Current colorimetric methods for quantitative determination of seed viability (SV) with 2,3,5-triphenyl tetrazolium chloride (TTC) have been plagued by issues of being cumbersome and time-consuming during the experimental process, slow in extraction and staining, and exhibiting inconsistent results. In this work, we introduced a new approach that combines TTC-staining with high-temperature extraction using dimethyl sulfoxide (DMSO). The optimization of the germination stage, TTC-staining method, and 1,3,5-triphenylformazan (TTF) extraction method were meticulously carried out as follows: When the majority of wheat seeds had grown the radicle, and the length of radicles was approximately equal to the seed length (24 h-germination), 2 g germinating seeds were placed into a beaker (20 mL) containing 5 mL 10 g·L-1 TTC solution. The seeds were stained with TTC in the dark at 25 °C for 1 h. Following the staining, 1 mL 1 mol·L-1 H2SO4 was added to stop the reaction for 5 min. The H2SO4 solution was then removed, and the seeds were gently rinsed with deionized water. Subsequently, the TTF produced in the seeds was extracted directly with 5 mL DMSO solution at 55 °C for 1 h. The absorbance of the extract was measured at 483 nm, and the index of SV was calculated according to a predetermined TTC calibration curve and expressed by mg TTC·g-1 (seed)·h-1. The new method has been demonstrated to be rapid, stable, and highly sensitive, as evidenced by the accurate measurement of seed viability with different aging degrees.
Keywords: 1,3,5-triphenylformazan (TTF); 2,3,5-triphenyl tetrazolium chloride (TTC); dimethyl sulfoxide (DMSO); extraction; germination stage; seed viability; staining; wheat seed.