**1. Introduction**

Plant cryopreservation involves the storage of plant tissues (usually seed or shoot tips) in liquid nitrogen (LN) at -196°C or in the vapour phase of LN at -135°C in such a way that the viability of stored tissues is retained following re-warming (Day et al., 2008; Hamilton et al., 2009). Cryopreservation is usually applied to species with recalcitrant (i.e. dehydration sensitive) seeds that are not storable by any other means, or preservation of specific cultivars of vegetatively propagated crop plants like banana or potato, or for unique ornamental genotypes (Halmagyi et al., 2004; Kaczmarczyk et al., 2011a; Panis et al., 2005). Another reason to utilise cryostorage is to conserve endangered plant species, particularly where seeds may be extremely scarce or of doubtful quality and/or the species is threatened with imminent extinction (Decruse et al., 1999; Mallon et al., 2008; Mandal & Dixit-Sharma, 2007; Paunescu, 2009; Sen-Rong & Ming-Hua, 2009; Touchell et al., 2002).

The main advantage of cryopreservation is that once material has been successfully cooled to LN temperatures, it can be conserved in principle indefinitely, because at these ultra-low temperatures no metabolic processes occur. Replenishing a small volume of LN weekly in cryo-dewars is the only on-going maintenance operation usually required in cryostorage. There are further advantages to this approach: the low costs of storage, minimal space requirements and reduced labour maintenance compared to living collections and even when compared to maintenance of tissue cultures at room temperature. Once in storage, there is no risk of new contamination by fungus or bacteria, and cryogenically stored material has been reported to retain genetic stability (Harding, 2004). Depending on the species, small cryopreserved samples may take several weeks to re-establish shoot cultures, and several months to a year may be required to produce micropropagated plants capable of transfer to soil under greenhouse conditions and (following weaning) into the field.

Shoot tips (containing the apical meristem) are the most commonly used plant material for cryostorage. The apical meristem is composed of small unvacuolated cells served with a relatively small vascular system. The organised structure of apical meristems generally results in direct shoot formation after re-warming, thereby maintaining the genetic integrity

Current Issues in Plant Cryopreservation 419

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 re-

Vitrification involves the treatment of tissues in a mixture of highly concentrated penetrating and non-penetrating CPAs applied at non-freezing temperatures, followed by rapid cooling in LN (Gonzalez-Arnao et al., 2008; Panis & Lambardi, 2005). The combination of high intracellular solute concentrations (due to dehydration and some CPA penetration) and rapid cooling prevents the nucleation of water and the formation of ice crystals, both intracellularly and extracellularly, thus promoting the vitrification of water (Kreck et al.,

For plants sensitive to direct exposure to vitrification solutions, due to dehydration intolerance and osmotic stresses, a loading step of 10-20 minutes can be incorporated prior to incubation within the CPA solution. This is done by incubation of the samples within a less toxic/concentrated CPA solution (a media containing 0.4 M sucrose and 2 M glycerol proved highly effective [Nishizawa et al., 1993; Sakai et al., 1990]), thereby improving dehydration tolerance. The CPAs used in vitrification usually contain high concentrations of glycerol, dimethyl sulfoxide (DMSO), ethylene glycol and various sugars (Day et al., 2008). The most commonly used mixture of CPAs for vitrification is plant vitrification solution 2 (PVS2), which consists of 30% (w/v) glycerol, 15% (w/v) ethylene glycol and 15% (w/v)

Exposure time to cryoprotective solutions is a vital step in the cryostorage process. Volk and Walters (2006) demonstrated that the extent of penetration of PVS2 into mint and garlic shoot tips was directly proportional to exposure time. The water content of the shoot tips also significantly decreased with an increase in exposure time to PVS2 (Volk & Walters, 2006). Greater penetration of CPAs can be useful as it helps to increase the internal solute concentration and may contribute to maintaining cell volume, thus preventing damage to the cells (Meryman, 1974). However, overexposure to CPAs may cause damage to the cells

The vitrification protocol is a more widely applied cryopreservation method than slow cooling due to its ease of use, high reproducibility and the wide range of species with complex tissue

DMSO in basal culture medium containing 0.4 M sucrose (Sakai et al., 1990, 2008).

owing to the toxic nature of the CPAs or excessive dehydration.

**2.2 Encapsulation-dehydration** 

warming (Panis & Lambardi, 2005).

2011; Mandumpal et al., 2011; Reinhoud et al., 2000).

**2.3 Vitrification** 

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 explants in plant cryopreservation (Reed, 2008).

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).
