**4.2 Sterols**

Sterols are steroid alcohols that are integral components of living cells. They are formed as intermediate molecules during the metabolic production of hormones and are an integral component of cell membranes (Hartmann, 1998; Hodzic et al., 2008). Sterols are non-polar molecules with a polar hydroxyl (-OH) side chain that allows them to interact with the polar and non-polar groups of the phospholipid bilayer. They restrict the motion of the fatty acid chains in phospholipids, thus controlling the fluidity of the cell membrane (Hartmann, 1998). The major plant sterols are cholesterol, campesterol, sitosterol and stigmasterol (Grunwald, 1975). Sitosterol is usually found in higher concentration in apex tissue than stigmasterol (Grunwald & Saunders, 1978). The composition and concentration of these sterols within the membrane modify its permeability and fluidity (Grunwald, 1975; Grunwald & Saunders, 1978; Nes, 1974). Cholesterol has the greatest stabilising effect on membranes due to its small side chain (Grunwald & Saunders, 1978).

The ability of sterols to alter stability and permeability of membranes can have a large effect on post-cryopreservation survival of plant tissue. The ratio of stigmasterol to sitosterol has been found to increase after sucrose preculture and was unfavourable to obtaining high percentages of shoot regeneration after cryopreservation of banana meristems (Zhu et al., 2006). Marsan et al. (1998) investigated the interactions of sitosterol and stigmasterol with phosphatidylcholine molecules in soybean and discovered, using neutron scattering experiments, that sitosterol has a greater effect than stigmasterol on the ordering of the fatty acyl chains of PC and increasing the hydrophobic thickness of PC bilayers. Cold acclimation of winter rye (*Secale cereale*) seedlings showed an increase in free sterol content, with β-sitosterol having the largest increase (Lynch & Steponkus, 1987). Uemura and Steponkus (1994) also found an increase of β-sitosterol with cold acclimation in winter rye; however, these results were not mimicked in spring oats (*Avena sativa* L. cv Ogle), where there was no significant change in β-sitosterol, but the stigmasterol proportion increased, whilst cholesterol decreased.

#### **4.3 Soluble sugars**

Intracellular soluble sugars and sugar alcohols, such as the ones used in preculture media, reduce damage sustained by cell membranes when the cells undergo desiccation and can

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

Phospholipids are amphiphilic molecules that form the core structure of the cell membrane lipid bilayer. They consist of a polar head (containing a phosphate group and simple organic molecule) and a (mostly) non-polar fatty acid tail. The most common phospholipid components of membranes include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS) and phosphatidic acid (PA). Phospholipids essentially maintain the structure, fluidity and permeability of the membrane, determined to some extent by the type of phospholipid present and the level of unsaturation in their fatty acid tails (Emmelot & Vanhoeven, 1975;

Sterols are steroid alcohols that are integral components of living cells. They are formed as intermediate molecules during the metabolic production of hormones and are an integral component of cell membranes (Hartmann, 1998; Hodzic et al., 2008). Sterols are non-polar molecules with a polar hydroxyl (-OH) side chain that allows them to interact with the polar and non-polar groups of the phospholipid bilayer. They restrict the motion of the fatty acid chains in phospholipids, thus controlling the fluidity of the cell membrane (Hartmann, 1998). The major plant sterols are cholesterol, campesterol, sitosterol and stigmasterol (Grunwald, 1975). Sitosterol is usually found in higher concentration in apex tissue than stigmasterol (Grunwald & Saunders, 1978). The composition and concentration of these sterols within the membrane modify its permeability and fluidity (Grunwald, 1975; Grunwald & Saunders, 1978; Nes, 1974). Cholesterol has the greatest stabilising effect on

The ability of sterols to alter stability and permeability of membranes can have a large effect on post-cryopreservation survival of plant tissue. The ratio of stigmasterol to sitosterol has been found to increase after sucrose preculture and was unfavourable to obtaining high percentages of shoot regeneration after cryopreservation of banana meristems (Zhu et al., 2006). Marsan et al. (1998) investigated the interactions of sitosterol and stigmasterol with phosphatidylcholine molecules in soybean and discovered, using neutron scattering experiments, that sitosterol has a greater effect than stigmasterol on the ordering of the fatty acyl chains of PC and increasing the hydrophobic thickness of PC bilayers. Cold acclimation of winter rye (*Secale cereale*) seedlings showed an increase in free sterol content, with β-sitosterol having the largest increase (Lynch & Steponkus, 1987). Uemura and Steponkus (1994) also found an increase of β-sitosterol with cold acclimation in winter rye; however, these results were not mimicked in spring oats (*Avena sativa* L. cv Ogle), where there was no significant change in β-sitosterol, but

Intracellular soluble sugars and sugar alcohols, such as the ones used in preculture media, reduce damage sustained by cell membranes when the cells undergo desiccation and can

membranes due to its small side chain (Grunwald & Saunders, 1978).

the stigmasterol proportion increased, whilst cholesterol decreased.

(Furt et al., 2011).

**4.1 Phospholipids** 

van Meer et al., 2008).

**4.3 Soluble sugars** 

**4.2 Sterols** 

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