Development of a Cryopreservation Procedure Using Aluminium Cryo-plates

A cryopreservation procedure using an aluminium cryo-plate was successfully developed using in vitro-grown Dalmatian chrysanthemum (Tanacetum cinerariifolium) shoot tips. Shoot cultures were cold-hardened at 5°C on MS medium containing 0.5 M sucrose over a period of 20 to 40 days. Shoot tips with basal plate (1.0-1.5 x 1.0 mm) were dissected from shoot cultures and precultured at 5°C for 2 days on MS medium containing 0.5 M sucrose. Precultured shoot tips were placed on aluminium cryo-plates (7 mm x 37 mm x 0.5 mm) with 10 wells (diameter 1.5 mm, depth 0.75 mm) and embedded in alginate gel. Osmoprotection was performed by immersing the cryo-plates for 30 or 60 min in 25 ml pipetting reservoirs filled with loading solution (2 M glycerol + 1.4 M sucrose). For dehydration, the loading solution was replaced with PVS 7M vitrification solution (30% (w/v) glycerol, 19.5% (w/v) ethylene glycol and 0.6 M sucrose in liquid MS basal medium), which was applied for 40 min. After rapid immersion in liquid nitrogen, shoot tips attached to the cryo-plates were rewarmed by immersion in cryotubes containing 2 ml 1 M sucrose solution. Using this procedure, regrowth of cryopreserved shoot tips of line 28v-75 reached 77%. This protocol was successfully applied to six additional lines, with high regrowth percentages ranging from 65% to 90%. By contrast, the modified vitrification protocol tested as a reference produced only moderate regrowth percentages. This new method displays many advantages and will facilitate large scale cryostorage in genebank.