Effect of cryopreservation on apple genetic resources at morphological, chromosomal, and molecular levels

Shoot tips of three apple genotypes, namely, Malus pumila cv. M26, Gala, and Hokkaido No. 9, were successfully cryopreserved using a modified encapsulation-dehydration method. As a result, in addition to a high survival rate and regeneration rate, the capacity of shoots regenerated from cryopreserved samples to root was enhanced. Eight M26 single-bud sibling lines were used to assess genetic stability. Although cytological examination revealed a ploidy difference in the noncryopreserved control, the ploidy constitution remained relatively stable during the period of cryopreservation. Amplified fragment length polymorphism (AFLP) assay was performed to detect DNA-level variation. No change in DNA fragment pattern and number was observed between the control and the cryopreserved samples. In addition, methylation-sensitive amplified polymorphism (MSAP) assay was carried out to investigate the DNA methylation status during the period of cryopreservation. It was found that cryopreservation induced a decrease in DNA methylation level.