**5. Genetic and epigenetic stability**

The aim of successful cryopreservation is to maintain genetically stable plant material. While cryopreservation is now recognised as the method of choice for the long term preservation of plant material, the usefulness of cryostorage only applies if it does not lead to genetic changes in the plant species of interest (Zarghami et al., 2008). It is thus recommended to avoid the use of tissue in a non-organised dedifferentiated state, such as callus, and to use organised tissue like shoot tips instead to reduce the likelihood of nondesirable genetic mutations (Benson et al., 1996; Harding, 2004) as well as due to their higher regrowth rates (Bunn et al., 2007). Cryopreservation can cause injury at cellular level, but it is not clear if this injury can change the genetic composition of plants. The genetic stability of cryopreserved plants has nonetheless been confirmed for most of the analysed samples at morphological, histological and molecular level (Harding, 2004). Where differences between control and cryopreserved genotypes were found, it was suggested that the genetic changes might not be associated with the cryopreservation process itself, but rather that they are caused by the overall tissue culture, cryoprotection and regeneration process (Harding, 2004).

Comparisons of morphological development and analyses of the characteristics of control and cryopreserved plants have shown no differences in many species, such as *Prunus*, sugarcane, onion, kiwi, *Eucalyptus*, coffee, *Dendrobium* and *Cosmos* (Harding, 2004), as well as in the hybrid aspen (*Populus tremula*), an economically important woody plant and widely used model forest plant (Jokipii et al., 2004). Alterations in phenotype related to flower colouring have been observed in *Chrysanthemum*, which might be related to the chimeric structure of the plant (Harding, 2004). Morphological and phenotypic studies in potato, where shoot tips were used for cryopreservation, showed stable genetic integrity (Kaczmarczyk et al., 2010). Biometric studies examining morphological characters, agronomic traits or vegetal development descriptors in *Dioscorea floribunda*, sugarcane and banana revealed no significant differences between cryopreserved-derived and control plants (Harding, 2004). No abnormalities in chromosome number or cell structure were observed in cryopreserved *Vanda pumila* (*Orchidaceae*), with the regrown shoot primordia being able to induce new meristematic tissues like those of the non-cryopreserved controls (Na & Kondo, 1996). Long term storage of cryopreserved plants, such as strawberry, pea, *Rubus* and potato, did not result in any overall changes in regeneration capability and phenotype when regenerated explants were compared at the time point of storage as well as 10, 12 and 28 years later (Castillo et al., 2010; Caswell & Kartha, 2009; Keller et al., 2006; Mix-Wagner et al., 2003).

Histological studies and cytological analysis using flow cytometry have confirmed the genetic stability of plant species such as pea, oil palm, silver birch, *Rubus*, *Solanum tuberosum* and rice (Harding, 2004). Biochemical analyses have compared products of gene expression such as the formation and concentration of secondary metabolites. Examples of compounds compared in cryopreserved and control plants have been diosgenin in *Dioscorea floribunda*, chlorophyll and pyrethrin in *Chrysanthemum*, hypericin production in *Hypericum perforatum*

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,

The aim of successful cryopreservation is to maintain genetically stable plant material. While cryopreservation is now recognised as the method of choice for the long term preservation of plant material, the usefulness of cryostorage only applies if it does not lead to genetic changes in the plant species of interest (Zarghami et al., 2008). It is thus recommended to avoid the use of tissue in a non-organised dedifferentiated state, such as callus, and to use organised tissue like shoot tips instead to reduce the likelihood of nondesirable genetic mutations (Benson et al., 1996; Harding, 2004) as well as due to their higher regrowth rates (Bunn et al., 2007). Cryopreservation can cause injury at cellular level, but it is not clear if this injury can change the genetic composition of plants. The genetic stability of cryopreserved plants has nonetheless been confirmed for most of the analysed samples at morphological, histological and molecular level (Harding, 2004). Where differences between control and cryopreserved genotypes were found, it was suggested that the genetic changes might not be associated with the cryopreservation process itself, but rather that they are caused by the overall tissue culture, cryoprotection and regeneration process (Harding,

Comparisons of morphological development and analyses of the characteristics of control and cryopreserved plants have shown no differences in many species, such as *Prunus*, sugarcane, onion, kiwi, *Eucalyptus*, coffee, *Dendrobium* and *Cosmos* (Harding, 2004), as well as in the hybrid aspen (*Populus tremula*), an economically important woody plant and widely used model forest plant (Jokipii et al., 2004). Alterations in phenotype related to flower colouring have been observed in *Chrysanthemum*, which might be related to the chimeric structure of the plant (Harding, 2004). Morphological and phenotypic studies in potato, where shoot tips were used for cryopreservation, showed stable genetic integrity (Kaczmarczyk et al., 2010). Biometric studies examining morphological characters, agronomic traits or vegetal development descriptors in *Dioscorea floribunda*, sugarcane and banana revealed no significant differences between cryopreserved-derived and control plants (Harding, 2004). No abnormalities in chromosome number or cell structure were observed in cryopreserved *Vanda pumila* (*Orchidaceae*), with the regrown shoot primordia being able to induce new meristematic tissues like those of the non-cryopreserved controls (Na & Kondo, 1996). Long term storage of cryopreserved plants, such as strawberry, pea, *Rubus* and potato, did not result in any overall changes in regeneration capability and phenotype when regenerated explants were compared at the time point of storage as well as 10, 12 and 28 years later (Castillo et al., 2010; Caswell & Kartha, 2009; Keller et al., 2006; Mix-

Histological studies and cytological analysis using flow cytometry have confirmed the genetic stability of plant species such as pea, oil palm, silver birch, *Rubus*, *Solanum tuberosum* and rice (Harding, 2004). Biochemical analyses have compared products of gene expression such as the formation and concentration of secondary metabolites. Examples of compounds compared in cryopreserved and control plants have been diosgenin in *Dioscorea floribunda*, chlorophyll and pyrethrin in *Chrysanthemum*, hypericin production in *Hypericum perforatum*

glycolipids and sphingolipids (Zhu et al., 2006).

**5. Genetic and epigenetic stability** 

2004).

Wagner et al., 2003).

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

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

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 cryoprotective agents (Harding, 2004).
