Cryopreservation of In Vitro Shoot Tips of Ulluco (Ullucus Tuberosus Cal.) Using D Cryo-plate Method

BACKGROUND: Maintenance of in vitro collections of ulluco (Ullucus tuberosus Cal.) is cumbersome and costly in an ex-situ genebank. An alternative method for long term preservation which is safe and cost-effective is required. OBJECTIVE: To apply a novel cryopreservation procedure using the cryo-plate system to improve the long-term conservation of ulluco. MATERIALS AND METHODS: Initially V and D cryo-plate methods were tested, subsequently the D cryo-plate method was selected for ulluco cryopreservation. The D cryo-plate procedures were optimized for post-LN regrowth procedures including cold-hardening, sucrose addition in alginate gel, and duration of LS treatment. Optimized procedures were tested with 11 ulluco lines. RESULTS: Shoot tips were isolated from cold-hardened shoots for 3-4 weeks at 5°C were excised to 1.0–1.5 mm long and 0.5 mm wide and precultured for 16h at 25°C on MS with 0.3 M sucrose. The shoot tips were attached on the cryo-plates by alginate gel with 0.4M sucrose. The cryo-plates with attached shoot tips were treated with 2.0 M glycerol and 1.0 M sucrose solution for 90 min at 25°C and dehydrated on filter paper in a Petri dish by air current flow at 25°C for 45 min before direct immersion in LN. This optimized procedure was applied to shoot tips of 11 ulluco lines, resulting regrowth ranging from 73% to 97%, with an average of 90% post-LN regrowth. CONCLUSION: D cryo-plate is a practical and simple procedure for cryo-storage of in vitro grown ulluco shoot tips in an ex situ genebank.