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

Brassica

seeds were selected as the experimental material

and the effect of 5

5′-dithiobis-(2-nitrobenzoic acid) (DTNB) on the freezing tolerance of hydrated

Brassica

seeds was investigated under the cooling rate of -1 ℃/min. The results showed that DTNB significantly improved the germination rate of

Brassica

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 th

e expression of cold stress-responsive genes

including ICE1 and CBF. In conclusion

DTNB can improve the freezing tolerance of

Brassica

seeds after rapid cooling through molecular and physiological regulation.