Cryopreservation of cold-acclimated mint (Mentha spp.) shoot tips using a simple vitrification protocol

Accessions of Mentha × piperita, M. × villosa, and M. spicata were evaluated for regrowth after cooling in liquid nitrogen using shoot tips from in-vitro grown plantlets and a simple vitrification protocol with aluminium foil as a carrier. The influences of plant preculture, loading solution and loading time and of the effects of the cryoprotectant PVS 2 on plant regrowth after re-warming were investigated. Nodal segments were cultivated at constant temperatures of 20 or 25°C or in alternating temperature regimes (25 / 15°C or 25 / −1°C). The illumination was always 16 h per day. The re-growth levels after re-warming were significantly higher in plants pre-cultured at 25°C / −1°C than in plants cultivated at 20 or 25°C or at 25°C / 15°C for all nine tested accessions. The mean re-growth levels increased from 36% at 20 °C to 69% at alternating temperatures, respectively. The maximum of plant re-growth after re-warming was 89%. A pre-culture at alternating temperatures of 25°C / 15°C did not increase the recovery of plants. Loading in sucrose solutions with different dehydration capacities did not alter the plant re-growth. Differences in the loading time between 20 min and 2 h were not important for re-growth either. No significant differences were found between freezing without and with PVS 2 droplets on the aluminium foil. Re-grown shoots rooted easily on the re-growth medium and plantlets were successfully transferred to soil.