**3.3 Antioxidants**

An antioxidant can be defined as any molecule that "delays, prevents or removes oxidative damage to a target molecule" (Halliwell & Gutteridge, 2007). Antioxidants can be classified into two major groups: enzymes that catalytically remove ROS, or sacrificial antioxidant molecules that are preferentially oxidised to protect more important molecules by quenching ROS (Halliwell & Gutteridge, 2007). The addition of exogenous antioxidants during cryopreservation has been shown to result in increased survival in some cases (Uchendu et al., 2010; Wang & Deng, 2004). It is possible that the addition of exogenous antioxidants may also aid in reducing oxidative stresses immediately following re-warming, when cellular metabolism has not been restored to its original state.

Glutathione (GSH) is one of the main water soluble sacrificial antioxidants in plants (Kranner et al., 2006). This low molecular weight thiol is converted to its oxidised form (GSSG) upon interaction with ROS. The ratio of reduced to oxidised GSH is a good indicator of oxidative stress, as an increased amount of GSSG indicates the inability of the plant to control oxidative damage, thereby triggering premature cell death (Kranner et al., 2006). GSH is recycled by the enzyme GSH reductase using NADPH as the electron donor (Halliwell & Gutteridge, 2007). Ascorbic acid is the most abundant water-soluble antioxidant in plant cells (Foyer & Noctor, 2009). This antioxidant is able to quench free radicals, forming the stable radical semidehydroascorbate, which can be further oxidised to dehydroascorbate by another free radical. *Arabidopsis* mutants, which were unable to express ascorbic acid activity, demonstrated the important role this antioxidant plays in reducing oxidative stress, as these plants were not viable following exposure to photooxidative stress (Dowdle et al., 2007). Tocopherol (Vitamin E) is a lipophilic antioxidant. This is the main antioxidant involved in membrane protection against lipid peroxidation, as it is preferentially oxidised instead of PUFAs (Halliwell & Gutteridge, 2007; Uchendu et al., 2010). This antioxidant is thought to be reduced to its original, functional state by ascorbic acid (Packer et al., 1979). Carotenoid pigments are the main defence in chloroplasts, where large amounts of singlet oxygen can be produced if the activated chlorophylls transfer their energy onto oxygen. The carotenoid pigments can absorb the energy directly from the

The use of DMSO as a free radical scavenger and probe for the hydroxyl radical can be utilised with VHS. The formation of methane when DMSO interacts with the hydroxyl radical can be measured if the sample is placed in an airtight container, with the levels of methane detected correlating with the formation of the hydroxyl radical. This technique has been used as a measurement of oxidative stress in multiple different plant species, such as rice, cocoa, *Daucus carota* and flax (Benson & Withers 1987; Benson et al., 1995; Obert et al., 2005; Fang et al., 2008). The production of methane is particularly strong during the initial phase of recovery, where it is predicted that antioxidant activity and production is reduced, resulting in increased ROS (Fang et al., 2008). The use of chelating agents to reduce the formation of hydroxyl radicals from the Fenton reaction has shown significant benefits in plant cryopreservation. Desferrioxamine is the most common chelating agent used. It binds to and reduces the amount of free iron. The addition of desferrioxamine to rice cells during cryopreservation showed a decreased recovery period after cryopreservation (Benson et al., 1995). Detection of methane formation was delayed in flax tissue when exposed to desferrioxamine (Obert et al., 2005), indicating a delayed production of hydroxyl radicals.

An antioxidant can be defined as any molecule that "delays, prevents or removes oxidative damage to a target molecule" (Halliwell & Gutteridge, 2007). Antioxidants can be classified into two major groups: enzymes that catalytically remove ROS, or sacrificial antioxidant molecules that are preferentially oxidised to protect more important molecules by quenching ROS (Halliwell & Gutteridge, 2007). The addition of exogenous antioxidants during cryopreservation has been shown to result in increased survival in some cases (Uchendu et al., 2010; Wang & Deng, 2004). It is possible that the addition of exogenous antioxidants may also aid in reducing oxidative stresses immediately following re-warming,

Glutathione (GSH) is one of the main water soluble sacrificial antioxidants in plants (Kranner et al., 2006). This low molecular weight thiol is converted to its oxidised form (GSSG) upon interaction with ROS. The ratio of reduced to oxidised GSH is a good indicator of oxidative stress, as an increased amount of GSSG indicates the inability of the plant to control oxidative damage, thereby triggering premature cell death (Kranner et al., 2006). GSH is recycled by the enzyme GSH reductase using NADPH as the electron donor (Halliwell & Gutteridge, 2007). Ascorbic acid is the most abundant water-soluble antioxidant in plant cells (Foyer & Noctor, 2009). This antioxidant is able to quench free radicals, forming the stable radical semidehydroascorbate, which can be further oxidised to dehydroascorbate by another free radical. *Arabidopsis* mutants, which were unable to express ascorbic acid activity, demonstrated the important role this antioxidant plays in reducing oxidative stress, as these plants were not viable following exposure to photooxidative stress (Dowdle et al., 2007). Tocopherol (Vitamin E) is a lipophilic antioxidant. This is the main antioxidant involved in membrane protection against lipid peroxidation, as it is preferentially oxidised instead of PUFAs (Halliwell & Gutteridge, 2007; Uchendu et al., 2010). This antioxidant is thought to be reduced to its original, functional state by ascorbic acid (Packer et al., 1979). Carotenoid pigments are the main defence in chloroplasts, where large amounts of singlet oxygen can be produced if the activated chlorophylls transfer their energy onto oxygen. The carotenoid pigments can absorb the energy directly from the

when cellular metabolism has not been restored to its original state.

**3.3 Antioxidants** 

chlorophylls, thus suppressing the formation of singlet oxygen, and they can also quench the singlet oxygen directly (Halliwell & Gutteridge, 2007).

The enzyme superoxide dismutase (SOD) catalytically removes the ROS superoxide, producing oxygen and hydrogen peroxide. The removal of superoxide is more important than the formation of hydrogen peroxide, as superoxide is a more reactive species, causing wider damage in the cells. There is potential for SOD to cause formation of ROS if levels of hydrogen peroxide are not controlled. SOD contains a metal cofactor that can cause Fenton reactions and the formation of the hydroxyl radical. Catalase is the main enzyme involved in removing hydrogen peroxide, resulting in the decomposition of hydrogen peroxide to water and oxygen. This enzyme is vital for the removal of hydrogen peroxide before it can damage the cell or be converted to the highly reactive hydroxyl radical through Fenton's reaction.
