**2.3 Vitrification**

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

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

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

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

Current Issues in Plant Cryopreservation 421

While achieving an optimum cryopreservation protocol that successfully avoids ice damage is important, there are various other factors that can affect post-cryogenic survival (Fig. 2). During the cryopreservation procedure plant tissues are susceptible to a variety of stresses,

Fig. 2. Main causes of damage to plant tissues during cooling and cryopreservation

oxygen-free radical species and reactive oxygen non-radical derivatives (Table 1).

The formation of reactive oxygen species (ROS) during cryopreservation can occur during the many steps involved in this process. For example, ROS formation has been detected in photooxidative stress during tissue culture, during excision of shoot apices, osmotic injury and desiccation following application of CPAs, as well as during the rapid changes in temperature when the samples are first cryostored in LN and then re-warmed (Benson & Bremner, 2004; Roach et al., 2008). ROS are highly reactive molecules that can cause a wide range of damage in cells. There is a large variety of molecules that are classified as ROS, some of which include

**Radicals Non-Radicals** 

Hydroxyl (OH•) Peroxynitrite (ONOO-)

Alkoxyl (RO•) Hypobromous acid (HOBr)

Carbonate (CO3•-) Ozone (O3) Carbon dioxide (CO2•-) Singlet oxygen (1Δg)

Table 1. Common reactive oxygen species (ROS) (Halliwell & Gutteridge, 2007).

•-) Hydrogen peroxide (H2O2)

Peroxynitrous acid (ONOOH) Hypochlorous acid (HOCl)

**3. Free radical and oxidative damage in plant cryopreservation** 

including oxidative stresses (Benson & Bremner, 2004).

(modified from [Turner, 2001]).

**3.1 Reactive oxygen species (ROS)** 

Superoxide (O2

Hydroperoxyl (OOH•) Peroxyl (ROO•)

Singlet oxygen (1Σg+)

structure (such as shoot tips and embryos) that have been successfully cryopreserved with this procedure (Takagi et al., 1997; Touchell et al., 1992; Vidal et al., 2005).
