Ascorbate deficiency compromises post-cryopreservation regeneration in Arabidopsis thaliana mutants: Antioxidant dynamics and oxidative DNA damage

Cryopreservation is important for the conservation of plant species. Oxidative stress, amongst the variety of stresses induced during the different steps of cryopreservation, can cause shifts in antioxidant contents that can lead to reduced post-cryopreservation survival and regrowth. A link between cryo-induced ascorbate deficiency and oxidative-induced DNA damage is not proven but could potentially contribute to poor regeneration of cryopreserved shoot tips. Accordingly, this study investigated cryo-tolerance (survival and regeneration of shoot tips), ascorbate and glutathione antioxidant content, and oxidative DNA damage, all at several steps of the cryopreservation protocol in wild type (WT) Arabidopsis thaliana shoot tips and two ascorbate deficient mutants, vtc2-1w and vtc2-4. Compared to the WT, the two A. thaliana mutants exhibited a significant decrease in post-cryopreservation regrowth and ascorbate levels but also exhibited higher initial glutathione content. Exogenous application of ascorbate resulted in a significant improvement in post-cryopreservation regrowth rates of the more susceptible vtc2-4 mutant shoot tips. Assessment of oxidative DNA damage showed that all treatments analysed exhibited detectable levels of 8-OHdG, indicating that some level of oxidative DNA damage occurs in both WT and mutant lines during cryopreservation; however, no correlation was discerned between increased 8-OHdG levels and observed lower ascorbate content in the mutants. These findings reveal that while exogenous ascorbate significantly improves post-cryopreservation regeneration in shoot tips of ascorbate-deficient mutants of A. thaliana, oxidative DNA damage was detectable throughout different steps of cryopreservation in WT and mutant plants, indicating that oxidative DNA damage was unlikely to be a significant contributor to the observed reduced regeneration rates in mutant plants.