**3.1 Reactive oxygen species (ROS)**

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


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

Current Issues in Plant Cryopreservation 423

therefore short-lived) and thus, direct measurement of the ROS present in cells is difficult due to time constraints, and does not reflect the damage that may be done prior to the ROS being quenched by antioxidants. Consequently, it is easier to measure the formation of byproducts of oxidative damage or the antioxidant status of the cells. The ratio of oxidised to reduced antioxidants is a good indication of ROS formation and the ability of cells to regulate oxidative stress. Identifying end products of ROS oxidation is an indication of the damage caused and is a sign that cells have been unable to satisfactorily quench ROS

The cell membrane represents one of the major areas where cryo-injury can occur. Any damage to the cell membrane can alter the delicate balance between intra and extracellular solutes, leading to cell death (Anchordoguy et al., 1987; Dowgert & Steponkus, 1984; Gordon-Kamm & Steponkus, 1984; Lynch & Steponkus, 1987). Lipid peroxidation of fatty acids (FA) in phospholipids can cause extensive damage to the cell membrane if the chain reaction is not controlled, leading to large areas where the semi-permeability of the membrane is altered and thus can no longer function normally (Benson et al., 1992; Halliwell & Gutteridge, 2007). Lipid peroxidation is caused when specific ROS (hydroxyl radical, peroxyl radical and singlet oxygen) interact with a FA. Polyunsaturated fatty acids (PUFA) are the most susceptible to peroxidation (Møller et al., 2007; Young & McEneny, 2001). Glutathione peroxidase can detoxify lipid peroxides by reducing them back to their lipid alcohol form and, in the process, oxidising glutathione (Benson, 1990). The formation of the toxic end-products of lipid peroxidation, such as malondialdehyde (MDA) and 4 hydroxylnonenal (HNE), can also cause damage to cells (Halliwell & Gutteridge, 2007). The toxicity of MDA is debatable (Halliwell & Gutteridge, 2007); however, there is evidence that MDA can interact with proteins and DNA, causing loss of function in proteins and mutations in DNA (Halliwell & Gutteridge, 2007; Hipkiss et al., 1997; Marnett, 1999). The formation of HNE has shown greater toxicity to cells as it can damage mitochondria, inhibit synthesis of DNA and proteins, and interfere with the action of repair proteins such as chaperones (Halliwell & Gutteridge, 2007). Identifying the formation of MDA and HNE is commonly used in cryopreservation as an indicator of oxidative stress. High levels of MDA or HNE detected have correlated with decreased survival rates in rice cell suspensions, olive somatic embryos, flax and blackberry shoot tips (Benson et al., 1992; Obert et al., 2005;

Volatile headspace sampling (VHS) measures the formation of volatile compounds released from a sample in a non-destructive and non-invasive assay method. This provides an important tool for measuring oxidative stress. Free radical damage can cause the formation of volatile compounds such as methane, ethane, ethylene and pentane. Quantification of these compounds can be indicative of oxidative damage. The detection of ethylene in plants is of particular importance as ethylene is a vital hormone. Fang et al. (2008) observed that decreased levels of ethylene production correlated with decreased survival and growth. The production of the other volatile compounds is indicative of lipid peroxidation, where increased levels correlate to excessive oxidative damage. Benson et al. (1987) observed a large increase in volatile compounds produced after thawing that has since been observed in

activity.

**3.2 Lipid peroxidation** 

Uchendu et al., 2010; Lynch et al., 2011).

other species.

Many of the more active ROS are free radicals, which are molecules that contain an unpaired electron, thus being able to react non-specifically with neighbouring molecules by removing electrons and causing a self-propagating chain reaction of radical formation. The removal of electrons can lead to a loss of function and structural alterations in macromolecules like proteins, lipids and DNA (Benson, 1990; Halliwell & Gutteridge, 2007). ROS are frequently produced as by-products during cellular metabolism. The electron transport chain used in respiration and photosynthesis are the major producers of ROS, caused by the leakage of free electrons onto molecular oxygen (O2), resulting in the formation of superoxide (Benson & Bremner, 2004; Benson, 1990; Halliwell & Gutteridge, 2007). The formation of ROS is controlled by a high concentration of antioxidants and proteins that can quench the ROS and fix the damage in these regions. Nevertheless, if there is a sudden increase in ROS formation then cellular repair processes can be overwhelmed and excessive damage can occur.

Temporarily reducing cryo-sample exposure to light immediately after cryopreservation has been shown to increase post-cryogenic survival due to the removal of photo-oxidative stress in the plants (Senula et al., 2007; Touchell et al., 2002). Photo-oxidative stress in plants can result in high levels of singlet oxygen (1Σg+) and superoxide (O2 •-) being produced, either from direct UV radiation on oxygen or the leakage of light energy onto oxygen from chlorophyll when the carotenoid pigments become saturated (Wise, 1995). Plants are highly susceptible to photooxidative stresses at low temperatures when exposed to strong light conditions. This can be demonstrated in alpine plants, many of which display adaptations (especially to leaves, i.e. the production of carotenoid pigments) that reduce photo-oxidation damage from enhanced UV-B radiation at high altitudes (Streb et al., 1998). When the ability of antioxidants to quench the formation of ROS and the recycling of antioxidants is reduced, greater damage occurs to the chloroplast through lipid peroxidation, inactivation of photosynthetic proteins and loss of pigments (i.e. bleaching) (Wise, 1995; Wise & Naylor, 1987). Damage to the chloroplast during chilling stress has been shown to severely impede growth rates (Partelli et al., 2009); therefore, reducing the damage that occurs to plant cells due to low temperature oxidative stress is vitally important for improving survival and recovery in cryopreservation.

The most reactive ROS commonly found in plants include superoxide (O2 •-), the hydroxyl radical (OH•), hydroperoxyl (OOH•) and singlet oxygen (1Σg+). Superoxide and singlet oxygen are often formed as by-products of the electron transport chain from both metabolism and photosynthesis, while hydroperoxyl and hydroxyl radicals are commonly formed in a process called Fenton's reaction, where hydrogen peroxide is converted into the hydroxyl or hydroperoxyl radical (1).

$$\rm Fe^{2+} + H\_2O\_2 \rightarrow Fe^{3+} + OH^\cdot + OH^\cdot$$

$$\rm Fe^{3+} + H\_2O\_2 \rightarrow Fe^{2+} + OOH^\cdot + H^\cdot \tag{1}$$

The formation of the hydroxyl radical is the major cause of lipid peroxidation in membranes, but can also cause a wide range of damage to all cellular components, including proteins and DNA (Halliwell & Gutteridge, 2007). The addition of specific chelating agents (such as desferrioxamine) has been shown to reduce the levels of iron in cryopreserved tissues, with subsequent decreased levels of the hydroxyl radical observed (Benson et al., 1995; Fleck et al., 2000; Obert et al., 2005).

Damage caused by ROS is difficult to quantify as these molecules are non-specific in their interactions, reacting freely with lipids, proteins and DNA. ROS are highly reactive (and therefore short-lived) and thus, direct measurement of the ROS present in cells is difficult due to time constraints, and does not reflect the damage that may be done prior to the ROS being quenched by antioxidants. Consequently, it is easier to measure the formation of byproducts of oxidative damage or the antioxidant status of the cells. The ratio of oxidised to reduced antioxidants is a good indication of ROS formation and the ability of cells to regulate oxidative stress. Identifying end products of ROS oxidation is an indication of the damage caused and is a sign that cells have been unable to satisfactorily quench ROS activity.

#### **3.2 Lipid peroxidation**

422 Current Frontiers in Cryobiology

Many of the more active ROS are free radicals, which are molecules that contain an unpaired electron, thus being able to react non-specifically with neighbouring molecules by removing electrons and causing a self-propagating chain reaction of radical formation. The removal of electrons can lead to a loss of function and structural alterations in macromolecules like proteins, lipids and DNA (Benson, 1990; Halliwell & Gutteridge, 2007). ROS are frequently produced as by-products during cellular metabolism. The electron transport chain used in respiration and photosynthesis are the major producers of ROS, caused by the leakage of free electrons onto molecular oxygen (O2), resulting in the formation of superoxide (Benson & Bremner, 2004; Benson, 1990; Halliwell & Gutteridge, 2007). The formation of ROS is controlled by a high concentration of antioxidants and proteins that can quench the ROS and fix the damage in these regions. Nevertheless, if there is a sudden increase in ROS formation

then cellular repair processes can be overwhelmed and excessive damage can occur.

vitally important for improving survival and recovery in cryopreservation. The most reactive ROS commonly found in plants include superoxide (O2

Fe2+ + H2O2 → Fe3+ + OH• + OH-

in high levels of singlet oxygen (1Σg+) and superoxide (O2

hydroxyl or hydroperoxyl radical (1).

1995; Fleck et al., 2000; Obert et al., 2005).

Temporarily reducing cryo-sample exposure to light immediately after cryopreservation has been shown to increase post-cryogenic survival due to the removal of photo-oxidative stress in the plants (Senula et al., 2007; Touchell et al., 2002). Photo-oxidative stress in plants can result

UV radiation on oxygen or the leakage of light energy onto oxygen from chlorophyll when the carotenoid pigments become saturated (Wise, 1995). Plants are highly susceptible to photooxidative stresses at low temperatures when exposed to strong light conditions. This can be demonstrated in alpine plants, many of which display adaptations (especially to leaves, i.e. the production of carotenoid pigments) that reduce photo-oxidation damage from enhanced UV-B radiation at high altitudes (Streb et al., 1998). When the ability of antioxidants to quench the formation of ROS and the recycling of antioxidants is reduced, greater damage occurs to the chloroplast through lipid peroxidation, inactivation of photosynthetic proteins and loss of pigments (i.e. bleaching) (Wise, 1995; Wise & Naylor, 1987). Damage to the chloroplast during chilling stress has been shown to severely impede growth rates (Partelli et al., 2009); therefore, reducing the damage that occurs to plant cells due to low temperature oxidative stress is

radical (OH•), hydroperoxyl (OOH•) and singlet oxygen (1Σg+). Superoxide and singlet oxygen are often formed as by-products of the electron transport chain from both metabolism and photosynthesis, while hydroperoxyl and hydroxyl radicals are commonly formed in a process called Fenton's reaction, where hydrogen peroxide is converted into the

 Fe3+ + H2O2 → Fe2+ + OOH• + H+ (1) The formation of the hydroxyl radical is the major cause of lipid peroxidation in membranes, but can also cause a wide range of damage to all cellular components, including proteins and DNA (Halliwell & Gutteridge, 2007). The addition of specific chelating agents (such as desferrioxamine) has been shown to reduce the levels of iron in cryopreserved tissues, with subsequent decreased levels of the hydroxyl radical observed (Benson et al.,

Damage caused by ROS is difficult to quantify as these molecules are non-specific in their interactions, reacting freely with lipids, proteins and DNA. ROS are highly reactive (and

•-) being produced, either from direct

•-), the hydroxyl

The cell membrane represents one of the major areas where cryo-injury can occur. Any damage to the cell membrane can alter the delicate balance between intra and extracellular solutes, leading to cell death (Anchordoguy et al., 1987; Dowgert & Steponkus, 1984; Gordon-Kamm & Steponkus, 1984; Lynch & Steponkus, 1987). Lipid peroxidation of fatty acids (FA) in phospholipids can cause extensive damage to the cell membrane if the chain reaction is not controlled, leading to large areas where the semi-permeability of the membrane is altered and thus can no longer function normally (Benson et al., 1992; Halliwell & Gutteridge, 2007). Lipid peroxidation is caused when specific ROS (hydroxyl radical, peroxyl radical and singlet oxygen) interact with a FA. Polyunsaturated fatty acids (PUFA) are the most susceptible to peroxidation (Møller et al., 2007; Young & McEneny, 2001). Glutathione peroxidase can detoxify lipid peroxides by reducing them back to their lipid alcohol form and, in the process, oxidising glutathione (Benson, 1990). The formation of the toxic end-products of lipid peroxidation, such as malondialdehyde (MDA) and 4 hydroxylnonenal (HNE), can also cause damage to cells (Halliwell & Gutteridge, 2007). The toxicity of MDA is debatable (Halliwell & Gutteridge, 2007); however, there is evidence that MDA can interact with proteins and DNA, causing loss of function in proteins and mutations in DNA (Halliwell & Gutteridge, 2007; Hipkiss et al., 1997; Marnett, 1999). The formation of HNE has shown greater toxicity to cells as it can damage mitochondria, inhibit synthesis of DNA and proteins, and interfere with the action of repair proteins such as chaperones (Halliwell & Gutteridge, 2007). Identifying the formation of MDA and HNE is commonly used in cryopreservation as an indicator of oxidative stress. High levels of MDA or HNE detected have correlated with decreased survival rates in rice cell suspensions, olive somatic embryos, flax and blackberry shoot tips (Benson et al., 1992; Obert et al., 2005; Uchendu et al., 2010; Lynch et al., 2011).

Volatile headspace sampling (VHS) measures the formation of volatile compounds released from a sample in a non-destructive and non-invasive assay method. This provides an important tool for measuring oxidative stress. Free radical damage can cause the formation of volatile compounds such as methane, ethane, ethylene and pentane. Quantification of these compounds can be indicative of oxidative damage. The detection of ethylene in plants is of particular importance as ethylene is a vital hormone. Fang et al. (2008) observed that decreased levels of ethylene production correlated with decreased survival and growth. The production of the other volatile compounds is indicative of lipid peroxidation, where increased levels correlate to excessive oxidative damage. Benson et al. (1987) observed a large increase in volatile compounds produced after thawing that has since been observed in other species.

Current Issues in Plant Cryopreservation 425

chlorophylls, thus suppressing the formation of singlet oxygen, and they can also quench

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.

Membrane systems within cells are usually the site of freezing injury in plants (Steponkus, 1984). Membrane stability is therefore important for reducing such injury. There are four types of injury: (i) expansion-induced lysis, where the cells overexpand as a result of increased extracellular osmotic pressure during warming/thawing; (ii) loss of osmotic responsiveness, where there is no osmotic change during warming due to a slow cooling rate (cells remain dehydrated); (iii) altered osmotic behaviour, where cells membranes turn "leaky", resulting in the release of water and solutes into the surroundings; and (iv) intracellular ice formation, where rapid cooling causes membrane disruption due to the

Fig. 3. Typical cell membrane structure consisting of a phospholipid bilayer with embedded sterols. Phospholipid chains are shown in grey, choline groups in blue and phosphate

The cell membrane is a bilayer consisting of different lipids and associated proteins (Fig. 3), where the lipids define the cell membrane structure and fluidity, and have a function in

groups in red, while sterol molecules are shown in yellow.

the singlet oxygen directly (Halliwell & Gutteridge, 2007).

**4. Plant cryopreservation and membrane structure** 

formation of ice crystals (Steponkus, 1984).

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.
