Update on long-term cryopreservation of almond germplasm

In 1999, we presented our work at XI GREMPA on conservation of almond (Prunus dulcis) germplasm by cryopreservation of almond shoot tips. "Nonpareil", "Ne Plus Ultra" and an almond-peach hybrid rootstock, Prunus dulcis "Titan" x P. persica "Nemaguard" were successfully cryopreserved for 6 months using a one-step vitrification technique. That work has since been extended to test for plant viability and genetic integrity after storage under liquid nitrogen for up to 2 years. Mean survival of shoot tips was 80% for "Ne Plus Ultra", 54% for "Nonpareil", and 78% for the hybrid rootstock, and there were no significant differences in survival between 3 days and 2 years. Genetic stability was tested by comparing DNA from the original trees, with leaves regrown from tissue culture, and from leaves regrown from cryopreserved shoot tips. Some changes in the structure and methylation of the DNA were found that were probably related to the in vitro culture process. These changes may have affected agronomic performance. Fruit and kernel characteristics have been monitored and compared to authentic non-preserved cultivars.