Chapter 1: Introduction 1
1.4. Desiccation Tolerance in Seeds 7
1.6.5. Cooling 24
Cooling of biological systems, no matter how rapid, will cause some ice crystallisation (Karow, 1969), unless all remaining intracellular water is non-freezable (Vertucci, 1990).
(The latter situation, however, is thought to be incompatible with viability retention of desiccation-sensitive embryos/embryonic axes [Pammenter et al., 1993; Pammenter and Berjak, 1999a].) The main challenge to cells during cooling is not just their ability to tolerate storage at cryogenic temperatures, but particularly to counteract the lethality of the intermediate temperatures (from -15 to -60°C) as they are cooled down to, and warmed back from, LN temperature (Mazur, 1984). These intermediate temperatures are known to favour ice formation and growth (Moor, 1973), which is regarded as the main source of cryo- damage. Also, according to Rice (1960), this temperature range allows for photophysical reactions that can result in the formation of free radicals and breaks in macromolecules as a
25 direct result of hits by background ionising radiation or cosmic rays. While such damage may be negligible, direct ‘hits’ can produce breaks in, or cause enough DNA damage, to become deleterious after re-warming to physiological temperatures, especially since no enzymatic repair can occur at such low temperatures (Mazur, 1984). Upon cooling, once the cells have passed through the intermediate temperature range, metabolic reactions will not persist, especially at LN temperatures, at which none of the thermally driven reactions can occur (Özkavukcu and Erdemli, 2002).
Cryoprotection and partial dehydration (employed individually or in combination) are therefore necessary in virtually all cases, to promote intracellular vitrification and avoid lethal intracellular ice crystallisation at sub-zero temperatures when cryopreserving hydrated plant tissues (Mycock et al., 1995). Ice crystallisation has been shown to be the predominant cause of intracellular damage during cryopreservation (Moor, 1973; Pearce, 2004; Sakai, 2004), but most contemporary cryobiologists agree that the damage acquired during cryopreservation is due to the effects of both desiccation (with its associated solution effects [Withers and King, 1979] and freezing (Kaviani, 2011). Cryoprotectants increase solute concentration inside the cell and decrease the amount of freezable water available intracellularly (Lovelock, 1953;
1954). Glycerol and DMSO are known to decrease the freezing point of water and many biological fluids by colligative action; glycerol decreases it to ~-46 ºC and DMSO decreases it to ~-73 ºC (Meryman et al., 1977). DMSO also protects the fluidity of membranes (Gurtovenko and Anwar, 2007a). Particularly, sugars (trehalose, sucrose) if present intracellularly, and glycerol as a CPA, protect against damage due to excessive water loss (Jochem and Korber, 1987).
Successful cryopreservation protocols entail optimisation of cooling rates in conjunction with explant-tissue hydration level, to eliminate – or at least minimise – nucleation of potentially lethal intracellular ice crystals. These factors are important determinants of cryopreservation success. Conventional cryopreservation protocols have mainly utilised slow (equilibrium) cooling rates (i.e. 0.5 to 2.0°C min-1 to ~-40°C [Kartha, 1985]), which favour extracellular ice nucleation and growth (Mazur, 1990) but allow for dehydration during cooling (Karow, 1969); however, the latter can, itself, be detrimental (Pritchard et al., 1995). Mazur (1963;
2004) showed that lethal intracellular freezing could be avoided if cooling was slow enough to permit sufficient water to leave the cell during progressive freezing of the extracellular
26 fluid (also known as equilibrium cooling). He also indicated that the rate of cooling differs between cells of differing size and water permeability, stating that a typical cooling rate around 1°C min-1 is appropriate for many mammalian cells after treatment with cryoprotectants such as glycerol or DMSO, but this rate is not a universal optimum.
However, it needs to be pointed out that these observations and recommendations come from work on cell suspensions (generally of animal origin), and not complex tissues such as embryos/axes excised from recalcitrant seeds.
Cooling rates that successfully minimise lethal crystallisation in plant cells still need to be empirically determined (Sershen et al., 2007), but in contrast to the arguments for equilibrium cooling, recent cryopreservation protocols for plant germplasm favour faster cooling rates (i.e. rates greater than 10 to hundreds of °C s-1, Walters et al., 2002b; Wesley-Smith et al., 1992; 2004a; b) that restrict the growth of intracellular ice crystals to non-lethal dimensions (Engelmann, 2004). Luyet et al. (1962) were the first to employ rapid (non-equilibrium) cooling rates (hundreds of °C min-1), and showed that intracellular ice formation could be restricted to below lethal levels. Faster cooling rates are also known to promote supercooling of the cell interior and to limit the extent of cellular dehydration incurred (Franks, 1985). As the cooling rates increase, ice crystals become more numerous but very small (Carrington et al., 1996), and may well be uniformly distributed intra- and extracellularly. The small intracellular crystals do have the potential to cause damage due to disruption to the cellular ultrastructure and can lead to cellular death (Karow, 1969); however, their impact on post- cryopreservation survival may be related to their localisation (Wesley-Smith, 2003). Based on their work on recalcitrant embryonic axes, Wesley-Smith and colleagues (2004a;b), also explain that at sufficiently low water contents (if achievable) the high intracellular viscosity slows ice crystallisation, making survival independent of cooling rate. They further highlight that at higher water contents, the reduced viscosity requires faster cooling to prevent ice crystal damage. The ability to cool such explants rapidly with increasing hydration therefore needs to be in balance with an increasing limitation to dissipate heat fast enough to prevent severe damage.
It is imperative that cryostored explants be rapidly re-hydrated. As with rapid cooling, this minimises the time during which explants pass through the temperature range which promotes ice crystallisation. Rapid rates of rehydration have been shown to result in higher
27 survival and normal onwards development of axes, compared with slow rehydration rates (Wesley-Smith et al., 2004a).