A simple and efficient protocol for cryopreservation of Taxodium hybrid ‘zhongshanshan’ embryogenic callus

Taxodium hybrid ‘zhongshanshan’ is a valuable tree species with significant ecological and economic importance. However, successful conservation of its germplasm remains challenging. In this study, we explored and established a viable method for cryopreservation of embryogenic callus of Taxodium hybrid ‘zhongshanshan’. In the pretreatment process, sucrose was identified as the optimal pretreatment agent, with a concentration of 0.5 M and a treatment duration of 36 h resulting in the fast recovery of callus viability. Dimethyl sulfoxide (DMSO) played a crucial role in preserving cell viability in the freezing process. The most favorable recovery effect was observed with a concentration of 10% DMSO, which led to the shortest recovery period (5 days) and a high callus proliferation rate of 4.30. In the recovery process, thawing at 35 °C enabled the callus to regain viability within 3 days and achieve a proliferation rate of 6.39. Notably, the recovered callus exhibited no discernible differences in appearance compared to normally proliferated embryogenic callus, and maintained the ability to differentiate into somatic embryos. This study provided valuable insights for the effective preservation of Taxodium hybrid ‘zhongshanshan’ genetic resources, which can also be utilized for long-term conservation.