**2. Plant cryopreservation methods**

In the last three decades a number of different cryopreservation protocols, such as classical slow-cooling, vitrification, droplet vitrification, encapsulation/dehydration and encapsulation/vitrification protocols have been developed and utilised for germplasm storage (Reed, 2008). The choice of cryopreservation method to attain the highest survival rates is largely dependent on the plant species and tissue type that is being cryostored.

#### **2.1 Slow cooling or controlled rate cooling**

This technique involves the simple dehydration of plant material before cryogenic storage in LN. This is can be done by slow cooling of the plant tissue to a temperature of approximately -40°C (Reinhoud et al., 2000). This forces the formation of extracellular ice ahead of intracellular ice, thus causing an outflow of water from the cells due to the resulting osmotic imbalance and, consequently, dehydration. Dehydration can also be brought about by incubation of tissue material on media containing a relatively high concentration of an osmoregulant, commonly sucrose, although other compounds can also be used (Panis et al., 2002). Usually water concentrations must be decreased to between 10% and 20% of the fresh weight for optimal cryogenic survival (Engelmann, 2004). This has the aforementioned effects of reducing the extent of ice crystal formation due to the reduced water concentration and assisting in the achievement of the vitrified state of water as a result of the increased solute concentration. These techniques do not necessarily make use of cryoprotective agents (CPAs), however they can be used in conjunction with them to further improve dehydration (Reinhoud et al., 2000), though these agents can be toxic to plant cells at high concentrations (Arakawa et al., 1990). Rapid re-warming rates are used after cryogenic storage to prevent ice crystal formation during thawing (Reinhoud et al., 2000). This approach can result in extreme rates of dehydration, which can cause cell volume reductions that are potentially lethal (Day et al., 2008). It has been suggested that slow-cooling is only suitable for non-organised tissues, as sufficient dehydration is more difficult to achieve in tissues with complex structures due to the different rates of water movement between and within plant cells with different characteristics (Gonzalez-Arnao et al., 2008).
