Cryopreservation of Shoot Tips of Tetraploid Potato (Solanum tuberosumL.) Clones by Vitrification

Abstract

In vitro-grown shoot tips of five tetraploid potato (Solanum tuberosumL.) clones were cryopreserved by vitrification. Excised shoot tips (0.5–0.7 mm) were pre-cultured on filter paper discs over half strength liquid Murashige and Skoog (MS) medium supplemented with 8.7 μmGA3and different combinations of sucrose (0.3, 0.5 and 0.7m) plus mannitol (0, 0.2 and 0.4m) for 2 d under a 16 h photoperiod at 24 °C. The pre-cultured shoot tips were either successively loaded with 20 and 60% PVS 2 solutions or directly exposed to concentrated vitrification solution before physical vitrification during liquid nitrogen treatment. The vitrified shoot tips were warmed rapidly and treated with dilution mixture (MS+1.2msucrose) for 30 min before plating on regrowth medium. Addition of mannitol to the pre-culture medium improved survival of vitrified shoot tips. Direct dehydration of pre-cultured shoot tips with concentrated PVS 2 was detrimental to survival of vitrified shoot tips. Shoot tips pre-cultured on medium containing 0.3msucrose plus 0.2mmannitol, and loaded with 20% PVS 2 for 30 min followed by 15 min incubation in 60% PVS 2 and 5 min incubation in 100% PVS 2 at 0 °C resulted in up to 54% survival after vitrification. About 50% of vitrified and warmed shoot tips formed shoots directly. Post-thaw culturing of vitrified shoot tips on medium containing an elevated level of sucrose (0.2m) under diffuse light for the first week enhanced the survival rate. Continuous culturing of vitrified shoot tips on high-sucrose medium induced multiple shoot formation.