# Cryobiotechnology of Plants: A Hot Topic Not Only for Gene Banks
## 2 Cryopreservation of Plants
### Plant Cryopreservation Methods
#### 2.1.2 Vitrification  
This approach is one of the most widely applied plant cryopreservation methods because it is relatively easy to carry out, no special equipment is needed, and it usually displays a high percentage of recovery. Vitrification is based on the formation of an amorphous glassy structure from intracellular solutes [1]. The plant material is treated with a highly concentrated vitrification solution that removes most or all freezable water from cells. The dehydrated material is then ultra-rapidly frozen by immersion in liquid nitrogen. The combination of dehydration and rapid freezing of cells causes residual water to solidify without crystallization, which could injure the living cells [17].  
Three main glycerol-based vitrification solutions (PVS1, PVS2, and PVS3) with different compositions have been reported. PVS1 consists of 19% (w/v) glycerol, 13% (w/v) ethylene glycol, 13% (w/v) propylene glycol, and 6% (w/v) DMSO dissolved in 0.5 M sorbitol. PVS2 consists of 30% (w/v) glycerol, 15% (w/v) ethylene glycol, and 15% (w/v) DMSO dissolved in 0.4 M sucrose. PVS3 consists of 50% (w/v) glycerol and 50% (w/v) sucrose dissolved in water [18]. In addition, two glycerol-free vitrification solutions were published in the past--the Towill cocktail (containing 35% ethylene glycol, 1 M DMSO, and 10% polyethylene glycol) and the Watanabe cocktail (containing 44.5% DMSO and 18.7% sorbitol) [19; 20].  
Among them, PVS2 is the most widespread, because of its less toxic effect on plant cells. Nevertheless, it cannot be used without any osmoprotection, because direct dehydration by PVS2  
Figure 1: Schematic diagram of major methods and steps of cryopreservation of shoot tips.  
causes damages to the cells and tissues of many plants. For successful cryopreservation, it is necessary to implement osmoprotection pretreatment with a loading solution (LS) containing 2 M glycerol and 0.4 M sucrose that induces osmo-tolerance and enables plants to achieve higher rates of recovery after cryopreservation [21; 22].  
Two main vitrification-based methods, namely encapsulation-vitrification and droplet vitrification, have been developed via the modification and optimization of vitrification techniques. The encapsulation-vitrification method was first reported by [PERSON] et al. (1995) [23] and combines encapsulation of plant germplasm within alginate beads and dehydration using a highly concentrated vitrification solution. The major merits of this mixed technique are better protection of encapsulated plant samples during vitrification and reduction of time needed for dehydration, compared to that of the classical encapsulation-dehydration method [22]. Using the method called droplet vitrification, shoot tips pretreated with LS and subsequently treated with vitrification solution are inserted individually into droplets of PVS2 that are placed on a strip of aluminum foil. The whole aluminum foil is then directly immersed in liquid nitrogen. The main benefit of this technique is the achievement of very high cooling/warming rates [24; 25].