Development of V and D cryo-plate methods as effective protocols for cryobanking

Abstract

Two efficient and simple cryopreservation methods using aluminium cryo-plates have been developed. In the V cryo-plate method, dehydration is performed using the vitrification solution PVS2, while in the D cryo-plate method, dehydration is achieved using the air current of the laminar flow cabinet or silica gel. To date, more than 20 papers have been published related to both methods. The main advantages of the V and D cryo-plate methods are as follows: handling of specimens throughout the procedure is easy and quick because only the cryo-plates are manipulated. The specimens attached on cryo-plates can be efficiently treated with loading solution (LS) and PVS2/air flow. Cooling and warming are performed easily by immersing the cryo-plates in LN and 1.0 M sucrose solution, respectively, resulting in ultra-rapid cooling and warming rates. High regeneration can be obtained using both methods. For species, which are sensitive to PVS2, the D cryo-plate method can be used. Both methods include preparation of material to be cryopreserved, preconditioning, excision, preculture, mounting of explants on cryo-plates, osmoprotection, dehydration by PVS or air flow, liquid nitrogen storage, rewarming and regeneration. Both protocols appear promising for cryopreservation of both herbaceous and woody plants including tropical plants after appropriate modifications of the procedures. Optimization of the dehydration time, preconditioning of materials and post-cryopreservation regrowth conditions are crucial to achieve high regrowth. These new cryopreservation methods will facilitate the efficient implementation of cryo-storage and long-term maintenance of plant genetic resources in genebanks.