Changes in Methylation and Structure of DNA from Almond Tissues during in vitro Culture and Cryopreservation

Abstract

Leaf explants were taken from mature leaves of two almond [Prunus dulcis (Miller) D.A. Webb] cultivars, Ne Plus Ultra and Nonpareil selection 15-1, and maintained in vitro to grow shoot tips. Shoot tips were grown also from a pre-existing in vitro culture of an almond-peach rootstock, P. dulcis `Titan' × P. persica `Nemaguard'. The shoot tips were harvested, cryopreserved, and tested for survival after 3 days and then at intervals of 3 months up to two years. The mean survival 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 24 months. The effects of in vitro culture and cryopreservation on DNA integrity were examined by both RAPD-PCR, and restriction enzyme digestion followed by RAPD-PCR, using DNA from the original trees from which the explants were derived, from leaves regrown from cultures that had undergone several passages of in vitro culture, and from leaves regrown from cryopreserved shoot tips. No detectable differences were found between the DNA fingerprints of each DNA sample using RAPD-PCR with seven different 10-mer primers. However, differences were detected when the DNA was first digested with the isoschizomeric pairs, Hpa II/Msp I and Bsp 143 I/Mbo I and then subjected to RAPD-PCR with six different 10-mer primers. Changes in the structure and methylation of DNA were found that were probably related to the process of in vitro culture, and in addition, methylation changes were detected that were probably associated with the cryopreservation process. These changes did not appear to be caused by the vitrification solution used before immersion of shoot tips in liquid nitrogen. While cryopreservation appears to be an ideal method for the long-term storage of almond germplasm, the significance of the alterations to both methylation and structure of DNA needs to monitored in regenerated plants, especially as they relate to agronomic performance when the regenerants become reproductively mature.