**2.2 Encapsulation-dehydration**

418 Current Frontiers in Cryobiology

of the resulting propagated material. While callus tissue (unorganised wound tissue) can also be cryostored, the risk of occurrence of genetic deviations may be higher when utilising the indirect organogenesis pathway. Besides shoot tips, callus cultures, cell cultures, somatic embryos, pollen or plant buds as well as recalcitrant and orthodox seeds can be used as

Plant cryopreservation began with research on the freezing of mulberry twigs in LN (Sakai, 1965). Since then, a huge variety of plants and genotypes have been successfully cryostored for conservation of agriculture and horticultural genotypes, as well as for endangered and threatened plant species (Gonzalez-Arnao et al., 2008; Hamilton et al., 2009; Kaczmarczyk et al., 2011b; Mycock et al., 1995; Reed, 2008; Sakai & Engelmann, 2007). This chapter reviews and gives examples of different plant cryopreservation protocols that have been successfully applied. It will focus on free radical damage and membrane structure, both important topics in the cryopreservation of biological tissues. The topic of genetic and epigenetic stability in plant cryopreservation is also discussed. Recent reviews of plant cryopreservation have been written by Benson (2008), Day & Stacey (2007), Hamilton et al. (2009) and Reed (2008).

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.

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

explants in plant cryopreservation (Reed, 2008).

**2. Plant cryopreservation methods** 

**2.1 Slow cooling or controlled rate cooling** 

(Gonzalez-Arnao et al., 2008).

This method, developed by Fabre and Dereuddre (1990), involves encapsulating shoot tips, somatic embryos or callus cells within alginate beads. This is followed by incubation in media with high sugar concentrations in order to raise intracellular solute concentrations and promote desiccation. Finally, silica gel or airflow is used to dehydrate the beads until the moisture content drops to 20-30%, before they are immersed in LN (Fabre & Dereuddre, 1990; Hamilton et al., 2009; Reinhoud et al., 2000). The encapsulating material is thought to promote a vitrified state in the tissue regardless of the cooling and re-warming rates, thus reducing damage from ice crystal formation (Scottez et al., 1992). Mechanical stress is also reduced because the bead protects the explants from damage during handling. The benefits of this method include avoiding the use of high concentrations of (potentially toxic) CPAs (Reinhoud et al., 2000) and the presence of a nutritive bead, which may enhance postregeneration survival or re-growth of the material following immersion in LN and rewarming (Panis & Lambardi, 2005).
