Development of PVS-Based Vitrification and Encapsulation–Vitrification Protocols

Cryopreservation is a very important tool for the long-term storage of plant genetic resources for future generations, requiring only a minimum of space and maintenance. With increasing interest in the genetic engineering of plants, the preservation of cultured cells and somatic embryos with unique attributes is assuming greater importance. Recently, cryopreservation was reported to offer real hope for enhancing the preservation of endangered and rare plants (Touchell 1995; Touchell and Dixon 1996). This chapter describes protocols for successful vitrification using plant vitrification solution 2 (PVS2), and highlights some of the factors contributing to high levels of post-LN recovery. The development of a simple and reliable method for cryopreservation would allow the widespread storage of cultured cells, meristems, and somatic embryos. Vitrification involving vitrification solutions (Langis et al. 1990; Sakai et al. 1990; Yamada et al. 1991) and encapsulation-dehydration techniques (Fabre and Dereuddre 1990) were developed in the 1990s, and the number of cryopreserved species has increased markedly since then (Sakai 1995, 1997; Engelmann and Takagi 2000). A vitrification procedure using an ethylene glycol-based vitrification solution and French straws was presented by Steponkus and colleagues. They reported successful cryopreservation of Dianthus and Chrysanthemum (Langis et al. 1990; Schnabel-Preikstas et al. 1992), and potato (Lu and Steponkus 1994). This chapter will outline the development and uses of PVS2 developed by Sakai and associates.