Programmed cell death and necrosis during cryopreparative drying of in vitro Eucalyptus grandis axillary buds

In preparation for cryopreservation, Eucalyptus grandis in vitro axillary buds were dried over silica gel. Pretreatment of the buds with 5 mg per L ABA resulted in partial resistance to water loss (0.76 to 0.45 g per g fresh mass basis) as compared with untreated buds (0.76 to 0.33 g per g) and was associated with the retention of viability (70 vs. 55%). The loss of viability of the dried buds was protracted over several days. Ultrastructural examination and vital staining demonstrated cellular and tissue responses to drying. The meristem appeared to withstand drying and 72 h of rehydration whilst the leaf primordia were destroyed immediately after drying. High reactive oxygen species (ROS) activity was associated with bud excision and drying. Caspase-3-like protease activity was detected after rehydration, thereby providing evidence that the dried buds, that had ultimately died, had undergone programmed cell death. ROS production is considered to be the trigger for programmed cell death.