Application of cryobanking for Platycodon grandiflorum in vitro axillary buds using cryo-plate methods

Abstract

The application of the V cryo-plate method using either PVS2 or PVS3 and the D cryo-plate method to Platycodon grandiflorum in vitro axillary buds was investigated. Precultured buds (1 mm) were attached to cryo-plates using calcium alginate gel. Then, osmoprotection treatment (2 M glycerol and 1 M sucrose) was performed by immersing the cryo-plates for 30 min at 25°C. The procedures for each cryogenic method were as follows: in the V cryo-plate method, the buds on cryo-plates were exposed to PVS2 for 40 min (highest regrowth 71.1%) or PVS3 for 50 min (highest regrowth 82.2%). In the D cryo-plate method, the buds on the cryo-plates were dehydrated under the air current of a laminar air flow cabinet for 60 min (highest regrowth 80.0%) at 25°C. Then, the cryo-plates were plunged directly into liquid nitrogen. After cryopreservation, buds on the cryo-plates were rewarmed and unloaded by immersion in 1 M sucrose solution for 15 min at 25°C for subsequent plant regeneration. The average regrowth seen on the V cryo-plates exposed to PVS3 and in the D cryo-plate method was higher than that of the V cryo-plates exposed to PVS2. The regrowth after cryopreservation in the V cryo-plate method with PVS2 was not stable. Thus, the V cryo-plate method with PVS3 and the D cryo-plate method are considered to be practical cryopreservation methods for P. grandiflorum germplasm.