title: Development of a cryopreservation procedure using aluminium cryo-plates.
abstract: 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 degree 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 degree 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 percent glycerol, 19.5 percent 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 degree. This protocol was successfully applied to six additional lines, with high regrowth percentages ranging from 65 to 90 percent. 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.
authors.affiliation: Genebank, National Institute of Agrobiological Sciences (NIAS), Tsukuba, Japan.
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Yamamoto
authors.forename: Tariq
authors.initials: T
authors.lastname: Rafique
authors.forename: Wickramage Saman
authors.initials: WS
authors.lastname: Priyantha
authors.forename: Kuniaki
authors.initials: K
authors.lastname: Fukui
authors.forename: Toshikazu
authors.initials: T
authors.lastname: Matsumoto
authors.forename: Takao
authors.initials: T
authors.lastname: Niino
mesh_terms: D000535:Aluminum; D018070:Biological Specimen Banks; D011723:Chrysanthemum cinerariifolium / Q000166:cytology* / Q000254:growth & development; D015925:Cryopreservation / Q000295:instrumentation* / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D055993:Germ Cells, Plant / Q000166:cytology / Q000254:growth & development; D018520:Plant Shoots / Q000166:cytology* / Q000254:growth & development; D058989:Vitrification
chemical_list: D003451:Cryoprotective Agents; D000535:Aluminum