Cryopreservation Protocols Using Aluminium Plates, V Cryo-plate and D Cryo-plate Method

Cryopreservation protocols using aluminium plates were successfully developed by our team. The size of an aluminum cryo-plate used is 7 mm × 37 mm × 0.5 mm with 10 to 16 wells fitting in 2 ml cryotube. The cryo-plates were custom-made by Taiyo Nippon Sanso Corp. Tokyo, Japan. The cryopreservation protocols developed using this cryo-plate are V cryo-plate method based on PVS2 dehydration of explants on the plates, and D cryo-plate method based on air dehydration. These procedures include the following steps: preparation of material to be cryopreserved, preconditioning, excision, preculture, mounting the shoot tips on cryo-plate, osmoprotection, dehydration, storage and regeneration. Both methods are efficient, ensures rapid cooling and warming rates, and reduce damage to explants before cryopreservation by attached shoot tips on cryo-plate. Also, they were developed with the aim of limiting the problems linked to operator skills and simplifying the procedure. The V cryo-plate method have been adapted to several plants, such as mint, dalmatian chrysanthemum, carnation, mulberry, mat rush and potato with high regrowth rates after cryopreservation. The D cryo-plate method have been adapted mat rush and mulberry. Both are a very practical cryopreservation methods now, appearing to be promising for the cryopreservation of other plants with a slight modification of the procedure. The V cryo-plate method and D cryo-plate method are complementary to each other. Cryopreservation using cryo-plate will facilitate large scale cryostorage of plant genetic resources with uniform and healthy materials. This research was partially supported by JST/JICA, SATREPS.