**4.4 Preconditioning and cold acclimation**

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

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, 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

Current Issues in Plant Cryopreservation 429

L. (Skyba et al., 2010; Urbanova et al., 2006), betalanin pigments in *Beta vulgaris* and nicotin alkaloids in *Nicotiana rustica*, which were all unchanged in cryopreserved plants and thus confirmed the integrity of metabolic functions after cryostorage (Harding, 2004). Similar stability was observed upon comparison of proteins and enzymes (Marin et al., 1993; Paulet

A variety of different techniques and markers have been applied to compare genomic DNA patterns, such as restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPDs) fragments, simple sequence repeat (SSR) analysis and amplified fragment length polymorphism (AFLP). Most studies have confirmed the presence of genetic stability (Castillo et al., 2010; Helliot et al., 2002) and where changes in the genome have been found, such as in sugarcane and potato with RFLP markers, the changes could not be related

In contrast to genetic variations manifested by DNA nucleotide sequence alterations, epigenetic changes do not change the original DNA sequence (Boyko & Kovalchuk, 2008) but can result in heritable changes of gene expression. Typical features of epigenetic characteristics are DNA methylation, histone modification and changes in chromatin structure (Boyko & Kovalchuk, 2008). Epigenetic gene regulatory mechanisms have a function in plant development and might be influenced or changed by environmental conditions and osmotic stress during tissue culture and cryopreservation. Some recent studies have analysed epigenetic characteristics like DNA methylation in tissue culture and cryopreserved plants. Modifications in DNA methylation have been found in almond (Channuntapipat et al., 2003), papaya (Kaity et al., 2008), chrysanthemum (Martín & González-Benito, 2006), *Ribes* (Johnston et al., 2009), strawberry (Hao et al., 2002a), citrus (Hao et al., 2002b) and potato (Kaczmarczyk et al., 2010). Changes in methylation might be caused by stressful *in vitro* conditions, osmotic dehydration and the application of

Many plant species have been successfully cryopreserved through the development of various cryopreservation methods. As a standard protocol, vitrification and droplet vitrification are widely applied. Shoot tips are the preferred material for cryostorage as they contain the meristem and an organised structure, with direct shoot development avoiding unstructured phases, which could lead to mutations. Preconditioning of plants (cold acclimation or sugar preculture) can have positive effects on survival and regeneration after cryopreservation, which could be due to increased membrane stability. Cryopreserved plants have been found to be genetically stable in most cases, but epigenetic changes have been detected, although most of the molecular analyses have only compared fractions of the

Success in cryopreservation cannot be guaranteed for all plants, as some species are recalcitrant to tissue culture or the cryopreservation process. Fundamental studies looking at membrane composition, membrane damage and repair are likely to help to elucidate why some species are cryosensitive and how cryopreservation protocols can be improved for

to the process of cryopreservation itself (Castillo et al., 2010; Harding, 2004).

et al., 1993; Wu et al., 2001).

cryoprotective agents (Harding, 2004).

**6. Conclusion** 

genome.

those species.

meristems prior to cryopreservation increased survival after warming and was related in most cases to a decrease of the double bond index in phospholipids, free fatty acids, glycolipids and sphingolipids (Zhu et al., 2006).
