Genetic stability analysis of chrysanthemum (Chrysanthemum x morifolium Ramat) after different stages of an encapsulation–dehydration cryopreservation protocol

Genetic stability in chrysanthemum (cultivar ‘Pasodoble’) apices was studied at each step of an encapsulation–dehydration cryopreservation protocol: control shoots (A), nodal segments after cold treatment (N), apices after osmotic stress (0.3 M sucrose) and cold treatment (P), encapsulation and culture in 0.8 M sucrose (S), dehydration (D), and cryopreservation (Cr). Two different markers were employed: RAPDs and AFLPs. Throughout the process, the origin of the apices (in vitro shoot from which they were excised) was recorded. Eight complete lines (from which DNA could be amplified after all the steps considered) were studied. Two out of twelve arbitrary primers showed polymorphisms. Three RAPD markers were replaced by three new ones in the Cr sample in one line. Using a different primer, a 700 bp fragment was absent from all samples from the 0.3 M sucrose-culture step (‘P’) onwards, in all the lines studied. The sequences of these fragments were studied to find similarities with known sequences. Polymorphic AFLP fragments were also observed, and most of the differences appeared from step ‘P’ onwards, pointing out the possible effect of this process (preculture on 0.3 M sucrose) in the DNA variation. These results show that genetic variation can appear throughout the cryopreservation process, and the low temperature itself is not the only stress risk of the technique. Therefore, genetic stability of the regenerants obtained after cryopreservation should be monitored.