Effect of cryopreservation on the structural and functional integrity of cell membranes of sugarcane (Saccharum sp.) embryogenic calluses

In this paper, we investigated if the differences consistently noted in survival and plantlet production between cryopreserved and non-cryopreserved, control sugarcane embryogenic calluses were related to modifications induced during cryopreservation in the structural and functional integrity of cell membranes. For this, the evolution of electrolyte leakage, lipid peroxidation products and cell membrane protein contents was measured during 5 d after cryopreservation. Differences between control and frozen calluses were observed only during the first 2 (electrolyte leakage) or 3 d (lipid peroxidation products and membrane protein content) after freezing. It was not possible to link these differences with the differences noted in survival and plant production between control and cryopreserved calluses. Additional studies are thus needed to elucidate which biochemical factors, linked to survival and plantlet regeneration, are affected by cryopreservation.

💡 Research Summary

This study investigated whether the reduced survival and plantlet regeneration observed in cryopreserved sugarcane (Saccharum sp.) embryogenic calluses could be linked to alterations in cell‑membrane structural and functional integrity caused by the freezing process. Embryogenic calluses derived from cv. CP52‑43 were cultured for 120 days, then subjected to a simplified freezing protocol: a 1‑hour pretreatment at 0 °C in a cryoprotective solution containing 10 % dimethyl‑sulfoxide (DMSO) and 0.5 M sucrose, cooling at 0.5 °C min⁻¹ to –40 °C, manual ice seeding at –10 °C, holding at –40 °C for 2 h, immersion in liquid nitrogen (–196 °C), and rapid thawing in a 40 °C water bath for 1 min. After thawing, calluses were placed on recovery medium and monitored for five days.

Three membrane‑related parameters were measured at 0, 1, 2, 3, 4 and 5 days post‑thaw: (1) electrolyte leakage (conductivity of imbibition water relative to total conductivity), (2) lipid‑peroxidation products (malondialdehyde, MDA, and a mixture of aldehydes) using the thiobarbituric‑acid reactive substances (TBARS) assay, and (3) total microsomal protein content via a Lowry‑Deoxycholate method. Each measurement was performed in triplicate.

Results showed that electrolyte leakage in cryopreserved calluses was markedly high immediately after thawing (≈70 % of total conductivity) but declined sharply, reaching levels comparable to non‑cryopreserved controls by day 3 and remaining low thereafter. This suggests that the freezing step caused only transient, reversible membrane permeability damage.

MDA and aldehyde concentrations peaked at 1–2 days in the cryopreserved samples (up to ~70 µmol g⁻¹ for MDA), whereas controls exhibited minimal changes. By day 4 both groups displayed similar low values (≈1–15 µmol g⁻¹). The early surge in lipid‑peroxidation products is consistent with a burst of reactive oxygen species (ROS) generated during freezing‑induced stress, leading to peroxidation of membrane lipids and formation of secondary toxic aldehydes.

Total microsomal protein content increased significantly in cryopreserved calluses, reaching a maximum on day 2 (approximately double the control value) before returning to baseline by day 4. This transient rise likely reflects synthesis of stress‑responsive proteins (e.g., dehydrins, heat‑shock proteins, membrane‑stabilizing peptides) that help protect membrane integrity under dehydration and low‑temperature conditions.

Despite the rapid normalization of these biochemical markers, long‑term outcomes differed markedly. Survival assessed 40 days after thawing was 90 % for cryopreserved calluses versus 100 % for controls, and plantlet regeneration measured 80 days after thawing was 150 plantlets per 500 mg fresh weight for cryopreserved samples compared with 270 plantlets for controls. Thus, the transient membrane disturbances could not explain the sustained reduction in viability and regeneration.

The authors discuss that ROS, rather than the measured lipid‑peroxidation products per se, may be the primary driver of lasting damage, as ROS can affect signaling pathways, gene expression, and metabolic homeostasis. They also note that mechanical injury from ice crystal formation and possible toxicity of the cryoprotectant (DMSO) could contribute to membrane destabilization. The observed decline in MDA and aldehydes after day 2 is attributed to activation of antioxidant defenses (β‑carotene, tocopherols, ascorbate, glutathione, and enzymes such as superoxide dismutase, catalase, and ascorbate peroxidase).

In conclusion, the study demonstrates that while electrolyte leakage, lipid peroxidation, and total membrane protein content recover within 3–4 days after cryopreservation, the overall survival and plantlet production remain compromised. This indicates that early oxidative stress and associated molecular responses have longer‑lasting effects on embryogenic competence. Future work should focus on quantifying antioxidant enzyme activities, profiling stress‑responsive proteins, and optimizing cryoprotectant composition and cooling rates to mitigate ROS generation and improve post‑thaw regeneration of sugarcane embryogenic calluses.