**4. Plant cryopreservation and membrane structure**

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 formation of ice crystals (Steponkus, 1984).

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

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

Current Issues in Plant Cryopreservation 427

improve membrane stability (Crowe et al., 1988; Steponkus, 1984). Smaller sugar molecules help membranes to osmotically retain water and may enter the interlamellar space to maintain a degree of hydration and increase the separation between membranes, thus reducing compressive stresses and, consequently, reducing the chance of a fluid-gel phase transition (Wolfe & Bryant, 1999). Furthermore, the polar hydroxyl (-OH) groups on sugars and sugar alcohols have been thought to interact with the polar membrane phospholipid headgroups and stabilise the membranes by maintaining the separation of the phospholipid molecules (Steponkus, 1984; Turner et al., 2001; Wolfe & Bryant, 1999). Turner et al. (2001) tested several sugars and sugar polyalcohols and determined that the small size of molecules such as glycerol and erythritol allowed them to 'pack' around membranes and have better bonding abilities. Additionally, the stereochemical arrangement of the -OH groups, particularly their orientation along one side of the molecules, imparted more stable hydrogen bonds with the membrane phospholipids and, consequently, resulted in more stable membranes (Turner et al., 2001). Nonetheless, recent biophysical studies have established that specific interactions of sugar molecules to phospholipid headgroups are not necessary to explain the stabilising effect of sugars on membrane gel to fluid transition temperatures (Lenne et al. 2006, 2007, 2009) and that sugars do not in fact insert between phospholipid headgroups but instead a solvation layer of water molecules separates them

Cold acclimation is the process in which plants being exposed to low non-freezing temperatures increase their freezing tolerance (Thomashow, 1999; Sakai & Engelmann, 2007). Preconditioning of raspberry and blueberry plants at 22/-1°C alternating temperatures for four weeks was essential for optimal post-cryopreservation shoot-tip regrowth using encapsulation-dehydration and vitrification protocols (Gupta & Reed, 2006). Similarly, better recovery rates were seen in mint (Senula et al., 2007) and yams (Leunufna &

Cold acclimation is thought to activate genes that improve plant survival at low temperatures by improving stability in membranes (Guy et al., 1985; Thomashow, 1999). Cell membrane phospholipids and sterols have been observed to increase in proportion upon cold acclimation in winter rye, with a particular increase in di-unsaturated PC and -sitosterol (Uemura & Steponkus, 1994). Changes in phospholipid and sterol composition were found in *Arabidopsis thaliana* after cold acclimation at 2°C for one week, which increased its freezing tolerance from -2°C down to -10°C (Uemura et al., 1995). Cell membrane phospholipid changes were also observed in oat leaves (Uemura & Steponkus,

Keller, 2005) when they were cold acclimated for several weeks before cryostorage.

1994). These changes may be related to improved membrane stability in these plants.

Preconditioning of plants has also been shown to increase antioxidant levels in plants prior to cryopreservation (Baek & Skinner, 2003; Dai et al., 2009; Harding et al., 2009; Zhao & Blumwald, 1998). Baek and Skinner (2003) analysed the expression of antioxidant genes in wheat species after cold acclimation and found increased expression of antioxidant enzymes, such as SOD and catalase. Increased levels of antioxidants may allow plants to better tolerate oxidative stress. Lynch and Steponkus (1987) observed an increase in the diunsaturated levels of PC and PE in winter rye seedlings. Sucrose pre-treatment of banana

from the phospholipid headgroups (Kent et al., 2010).

**4.4 Preconditioning and cold acclimation** 

signal transduction (Foubert et al., 2007; Furt et al., 2011). The three main classes of lipids found in cell membranes are glycerolipids (mostly phospholipids), sterols and sphingolipids (Furt et al., 2011).
