Development of – 80 °C storage for Allium shoot tips using D cryo-plate method

Abstract

D cryo-plate method was successfully adapted for storage at – 80 °C and – 196 °C of Allium shoot tips. The optimal D cryo-plate method using garlic shoot tips for the storage at – 80 °C and – 196 °C includes preculture of shoot tips for 2 days at 25 °C on solidified 1/2 MS medium containing 0.3 M sucrose, then placing them in wells on an aluminum cryo-plate, embedded in calcium alginate gel. Osmoprotection was performed by immersing the cryo-plates with shoot tips for 30 min at 25 °C in 2.0 M glycerol and 1.0 M sucrose. The optimal dehydration time of shoot tips was for 30 to 180 min at 25 °C. Cooling was performed by transferring the samples on the cryo-plate into 2 ml cryotubes, then cooling into a deep freezer at – 80 °C for 14 days or directly plunging into liquid nitrogen (− 196 °C) for 60 min. The regrowth rate of shoot tips stored both at – 80 °C and – 196 °C was almost 100%. This optimized procedure was applied to seven Allium spp. The average regrowth rates after cooling at – 80 °C and – 196 °C were 95.3 and 94.0%, respectively. Thus, the storage of shoot tips at – 80 °C using D cryo-plate method is considered to be efficiently applicable for practical genebank storage. D cryo-plate method was successfully adapted for storage at – 80 °C of Allium shoot tips. Grass transition temperature of shoot tips after air desiccation were − 39.4 °C.