Development of Encapsulation Dehydration

The application of cryopreservation to plants is relatively recent as the first report of successful cryopreservation was published by Sakai in 1960 with silver birch twigs, and in-vitro cultured flax cells were frozen by Quatrano in 1968. The first protocols developed in the 1980s included pre-treatment with cryoprotectants followed by controlled rate cooling. These protocols were based on freeze-induced dehydration (Sakai 1985; Kartha and Engelmann 1994; Engelmann 1997). Such protocols were applied to numerous species, especially from temperate origin; however, there were cases, particularly for plants of tropical origin, where such controlled cooling protocols did not produce good results (Bagniol et al. 1992; Haskins and Kartha 1980). Further research was thus carried out and at the beginning of the 1990s a set of new, vitrification-based protocols became available (Engelmann 2000, 2003). Vitrification can be defined as the transition of water directly from the liquid phase into an amorphous phase or glass, while avoiding the formation of crystalline ice (Fahy et al. 1984). Among these vitrification techniques a new technique termed encapsulation dehydration was developed for cryopreservation of pear and potato shoot-tips (Dereuddre et al. 1990; Fabre and Dereuddre 1990). This method is based on the technology developed for producing synthetic seeds, i.e. the encapsulation of explants in calcium alginate beads (Redenbaugh et al. 1986). Encapsulated explants are then precultured in liquid medium with a high sucrose concentration and partially desiccated before exposure to liquid nitrogen (LN). Encapsulating the explants allows exposure to extreme treatments including preculture with high sucrose concentrations and desiccation to low moisture contents (MCs) that would be highly damaging or lethal to non-encapsulated samples. Due to the extreme desiccation of explants, most or all freezable water is removed from cells, and vitrification of internal solutes takes place during rapid exposure to LN, thus avoiding lethal intracellular ice crystallization (Engelmann 1997). As a consequence, the whole or a large part of the frozen explant is kept intact after rewarming, which results in high survival, rapid and direct regrowth and reproducible results after cryopreservation (Engelmann 2000).