Determining the effect of pretreatments on freeze resistance and survival of cryopreserved temperate fruit tree dormant buds.

Freeze resistance is critical to successful dormant bud (DB) cryopreservation, and is affected by genotype, environmental conditions, dormancy phase and processing techniques. Pretreatment induced freeze resistance may contribute to more successful and efficient protocols for cryopreserving DB. Differential thermal analysis (DTA) was used to quantify the effects of cryopreservation pretreatments on freeze resistance of dormant budwood. Low temperature exotherm (LTE) profiles created by DTA could rapidly identify pretreatments that are contributing to increased freeze resistance in tree fruit species. In this study, DTA was used to help elucidate the effects of varying pretreatments (sucrose, desiccation and their combination) on peach, a model crop in tree fruit physiology that has shown little cryosurvival using the DB method in the past. Post cryopreservation recovery trials using an antimicrobial forced bud development (AFBD) protocol evaluated the ability of selected pretreatments, that improved freeze resistance based on DTA, to improve recovery of dormant budwood of various deciduous tree fruit and nut species. Precryogenic exposure to sucrose solution (5.0 M, 96 h), desiccation to 30% moisture content (MC) and their combination tested for their efficacy on improving postcryogenic viability in peach, apricot, sweet cherry, little walnut, black walnut, English walnut, apple, and pear. Among the different pretreatments tested, desiccation to 30% MC had the greatest impact on increasing freeze resistance and cryosurvival across most fruit species tested and little walnut. Gradual reduction of MC (from 40 to 25%) levels increased freeze resistance in peach (R=0.95) and increased some recovery outcomes (leaf, shoot and bud swell), however, this was not correlated with equal cryorecovery outcomes as severe bud cracking was observed. Overall, our approach linking freeze resistance and preconditioning treatments could help establish efficient species-specific cryopreservation protocols for a number of important temperate woody crops which could be recovered as complete plants by coupling AFBD and plant tissue culture.