Genetic and epigenetic stability of recovered mint apices after several steps of a cryopreservation protocol by encapsulation-dehydration. A new approach for epigenetic analysis.

The genetic and epigenetic stability (analysis of DNA methylation using MSAP markers) of mint (Mentha x piperita L.) apices was studied after each step of a cryopreservation protocol, by encapsulation-dehydration. The effect of the addition of an antioxidant (ascorbic acid) during one of the protocol steps was also evaluated. Eight-week old in vitro recovered shoots from apices after each step of the protocol were genetically stable when compared to control in vitro shoots, using RAPD and AFLP markers. The addition of ascorbic acid in the medium with the highest sucrose concentration did not improve recovery and did not have any effect on stability. Apices sampled immediately after each step showed increased epigenetic differences as the protocol advanced, compared to in vitro control apices, in particular related to de novo methylation events. However, after one-day in vitro recovery, methylation status was similar to control apices. To improve the quality of methylation data interpretation, a simple and fast method for MSAP markers analysis, based on R programming, has been developed which allows the statistical comparison of treatments to control samples and its graphical representation.