Effect of DTNB on Freezing Tolerance of Hydrated <italic>Brassica oleracea</italic> Seeds during Fast Cooling

The moisture content of seeds poses a significant challenge in their cryopreservation. Previous studies have shown that the hydrolysis of seed globulins can improve the freezing resistance of hydrated seeds. In this study, <italic>Brassica</italic> seeds were selected as the experimental material, and the effect of 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) on the freezing tolerance of hydrated <italic>Brassica</italic> seeds was investigated under the cooling rate of -1 ℃/min. The results showed that DTNB significantly improved the germination rate of <italic>Brassica</italic> seeds after rapid cooling. The initial temperature of ice crystal formation in seeds was delayed in DTNB treatment (control group vs. DTNB group: -14.78 ℃ vs. -16.43 ℃). Compared with the control, two different protein spots accumulated below 25 kDa; which were identified as 12 s storage globulins by mass spectrometry. The sulfhydryl group content, globulin, and its hydrolyzed polypeptide were significantly increased in the DTNB treatment. The non-enzymatic oxidants AsA and GSH increased by 1.26 times and 1.22 times, respectively, relative to the control. DTNB also promoted the expression of cold stress-responsive genes, including ICE1 and CBF. In conclusion, DTNB can improve the freezing tolerance of <italic>Brassica</italic> seeds after rapid cooling through molecular and physiological regulation.