**2.1 General principles**

Public and private efforts have been made to protect and conserve germplasm by preserving the genetic material of selected genotypes (e.g., Engelmann, 2000). As in other species, cork oak germplasm preservation can be done in situ (in the field and in natural environment),

and growth regulators type (Fernandes, 2011) confirming that cell cycle dynamics during somatic embryogenesis suffers exogenously-induced alterations, and in particular, it is conditioned by growth regulators (Gahan, 2007). We also found that using the two different somatic embryogenesis protocols available (Fernandes, 2011; Pinto et al., 2002), not only cell cycle dynamics changed with time during the process, but also genotypes with different somatic embryogenic competences had different cell cycle dynamics (Fernandes, 2011). Curiously, responsive genotypes showed cell cycles with similar progression profiles

Considering the key players regulating cell cycle dynamics, cyclins are among the most important. In a broad sense, D-type cyclins are thought to regulate the G1-to-S transition, Atype cyclins, the S-to-M phase control, and B-type cyclins regulate both the G2-to-M transition and intra-M-phase control (Gahan, 2007). The cyclin D (CYCD)/retinoblastoma pathway is believed to be involved in controlling both the commitment of cells to the mitotic cell cycle and decisions involving cell growth, differentiation, and cell cycle exit (Dissmeyer et al., 2009; Cools & Veylder, 2009). Key genes for growth and cell division are regulated by E2F transcription factors, which are inactive when bound by retinoblastoma. The phosphorylation of retinoblastoma is initiated by CYCD-containing cyclin-dependent kinases (CDKs) and is completed by cyclin E–CDK2, resulting in the dissociation of retinoblastoma from E2F factors, triggering the passage of cells from G1- to S-phase (Gahan, 2007). This key role for the G1 exit pathway results in it being the primary and predominant cell cycle control point. However, cyclin E–CDK2 is rate-limiting for entry into S-phase and can trigger S-phase in the absence of RB phosphorylation. CYCD3;1 are the best studied examples and expression of their genes is regulated by extrinsic signals, such as sucrose availability. CYCD3;1 expression is also regulated by plant hormones (Dewitte et al., 2003;

The E2F family plays a critical role in organizing cell cycle progression by coordinating early cell cycle events with the transcription of genes required for entry into S-phase (e.g., Inzé, 2000). Two major classes of genes possess characteristic E2F binding sites, the first class encodes essential enzymes in the pathways for nucleotide and DNA synthesis that are coordinately up-regulated in late G1. The second class corresponds to genes for regulators of cell cycle progression. Genes from both classes respond to ectopic expression of E2Fs from the first sub-group, namely those that can induce entry into S-phase (Dewitte & Murray,

The cell cycle involves a complex network of regulating molecules. So the control of all these classes of checkpoints regulators is under study in cork oak embryogenic (EC) and nonembryogenic (NEC) tissues (Santos, 2011, unpublished data). Understanding and controlling these checkpoints will become a powerful tool to both better understand the embryogenic

Public and private efforts have been made to protect and conserve germplasm by preserving the genetic material of selected genotypes (e.g., Engelmann, 2000). As in other species, cork oak germplasm preservation can be done in situ (in the field and in natural environment),

per se and to manipulate the developmental stages of embryogenic process.

(Fernandes, 2011).

for review see Dewitte & Murray, 2003).

2003; Gahan et al., 2007).

**2. Cryopreservation 2.1 General principles** 

which demands large areas and is susceptible to environmental hazards. Alternatively preservation may be done ex situ (for general review see Li & Pritchard et al., 2009). In particular, in vitro preservation allows that in a small area, large amounts of genotypes are multiplied and maintained under controlled conditions where environmental influences are minimal. However, precocious ageing as well as somaclonal variation and genetic instability may arise after long term culture (Brito et al., 2009).

Alternatively, cryopreservation is the storage of living materials at extremely low temperatures using usually liquid nitrogen (−196 °C), and is an ideal strategy for plant germplasm preservation (Benson, 2008; Feng et al., 2011; Wang & Perl, 2006). This preservation strategy allows not only the preservation of material in small volumes (involving low maintenance requirements) but also, by reducing to residual values the cell metabolism, it allows that cells are stored for long periods, with low probability of genetic instability occurrence (Feng et al., 2011). Cryopreservation therefore allows: the conservation of plant material minimizing occurrences of genetic instability, contaminations and diseases; the preservation of endangered, rare or selected genotypes. Cryopreservation is already being applied to several plant species including forest woody species (e.g., Sakai et al., 2008). Also different plant materials have been used in this preservation strategy: shoot tips, cell cultures, embryos and seeds (Feng et al., 2011).

For cryopreservation to be useful in breeding programs, it is necessary to develop the cryogenic technique *per se*, and to ensure that robust and efficient regeneration protocols are available. Freezing and thawing stages require that cells are structurally and functionally cryoprotected. This may happen naturally (e.g. some naturally dehydrated material) but usually it is induced artificially with treatment with cryoprotectants that influence ice formation and activity of electrolytes present in the solution. Ideally, cryoprotectants should have low or no cytotoxicity. Cryoprotectants may be: a) permeating compounds, such as dimethylsulphoxide (DMSO, used usually in the range of 5-10%) that has a rapid entrance rate and so requires short incubation periods; another permeating compound is glycerol (used often in the range of 10-20%); b) non-permeating compounds such as sugars, sugar alcohols, polyethylene glycol (PEG). Often mixtures of cryoprotectant compounds are preferred to improve their efficacy (e.g., combinations of PEG : glucose : DMSO). Finally other strategies as cold hardening or ABA treatment may increase the freezing resistance and survival rates of cells.

Plant cryopreservation strategies may include slow or rapid freezing approaches. The first is based on physico-chemical changes during the process, namely associated with apoplastic ice crystal formation while cytoplasm may remain free from intra-cellular ice formation. Slow freezing decreases therefore the osmotic potential of the cytoplasm contributing to the cell desiccation. Rapid freezing is achieved by immersion of the cryoprotectant-treated samples in N2, leading to an ultra-fast cooling that prevents the formation of ice crystals inside the cell (e.g., Sakai et al., 2008).

Some variants involve vitrification that includes a cell dehydration step prior to storage in N2 (Sakai et al., 2008). This may rely on the ability of concentrated solutions of cryoprotectants to become viscous to very low temperatures, without ice formation. Consequently, during the vitrification process plant cells are dehydrated and the cytoplasm vitrified during freezing, which allows that ice crystals are rarely formed. Vitrification has already been applied to a large number of species (Panis et al., 2005; Sakai et al., 2008).

Somatic Embryogenesis and Cryopreservation in Forest Species: The Cork Oak Case Study 405

a b

cryopreservation used CRY25 and CRY35, corresponding to the final % content of water). b) Acclimatized plant obtained from cryopreserved somatic embryos that were recovered and cultivated on MSWH for maturation and conversion (adapted from Fernandes et al., 2008). In this standard protocol for cork oak, somatic embryos or embryogenic clusters derived from mature trees and previously maintained on MS medium without growth regulators (MSWH) are used as samples. For encapsulation, embryos/clusters were separated and loaded in the alginate plus CaCl2 solutions, forming the beads (3–4 mm), each one contained one embryo/cluster. They were then pre-cultivated on sucrose-enriched standard liquid medium (MSWH with 0.7M sucrose) for 3 days. Beads were then desiccated by drying in the airflow of a laminar flow cabinet, and carefully weight loss was monitored for water content calculation. Two final water content (WC) values were assessed: 25% (CRY25) and 35% (CRY35). Afterwards, beads were placed in cryotubes (10 per vial), and immersed in N2 for 24 h. Samples' thawing was done by incubating the cvryotubes at 38 ºC (2 min) and incubating in detoxification solution (1 h). Beads were transferred to solid standard medium (MSWH) for regeneration (Figure 4a, b; Fernandes et

This cryopreservation technique developed by Fernandes et al. (2008) for cork oak somatic embryos is simple, effective and non-toxic for the species. Also, survival rates in encapsulated-dehydrated (but non-frozen) cork oak samples achieved 90%. We also demonstrated that cryopreserved somatic embryo derived clones were able to recovery,

In vitro regenerated plants may exhibit somaclonal variation as a result from genetic or epigenetic modifications (Fourré et al., 1997; Isabel et al., 1996). It is generally accepted that morphological, cytological and molecular variations may be generated by the imposed stress during in vitro or cryopreservation processes. These induced variations are conditioned by the genotypes used and/or by the techniques/protocols used. Theoretically,

leading to plants morphologically normal and that had genetic stability.

**3. Screening of genetic variation in cloned and cryopreserved material** 

Fig. 4. a) Percentage of survival of cork oak cells after 10 weeks (for two protocols of

al., 2008).

Nonetheless, during this process, usually complex and toxic solutions with high osmotic potential, such as the PVS2, are used for cryoprotection (Fernandes et al., 2008). Also the duration of the successive steps of a vitrification protocol is in general very short, hampering the simultaneous treatment of a large number of samples.

Some alternatives to vitrification-based techniques were developed, namely encapsulationdehydration strategies (e.g., Engelmann et al., 2008). Encapsulation-dehydration is based on concepts related with artificial seeds. Concisely, plant tissues, such as shoot tips or somatic embryos, are covered by for example alginate. Then, they are dehydrated (using exposures to highly concentrated solutions, and/or to air in a flow chamber), before being transferred to N2. This strategy has the advantage of using less toxic compounds such as glycerol than in other vitrification methods, thus minimizing stress conditions (e.g. Volk et al., 2006). It also has the advantage of easy and inexpensive manipulation, not requiring expensive instruments, as occurs in controlled freezing (Fernandes et al., 2008).

This method has been applied to many species, such as, for example, mulberry (Niino & Sakai, 1992), *Prunus* sp. (Brison et al., 1992), sweet potato (Feng et al., 2011; Hirai & Sakai, 2003), persimmon (Matsumoto et al., 2001), apple (Niino & Sakai, 1992; Paul et al., 2000), lily (Bouman & Klerk, 1990) and even grapevine embryogenic cell suspensions (Wang & Perl, 2006) or pear (Niino & Sakai, 1992; Scottez et al., 1992). In several assays as in those with *Robinia pseudoacacia*, it was demonstrated that encapsulation-dehydration originated better results than vitrification (Verleysen et al., 2005). Recently we have also demonstrated that encapsulation-dehydration was the most efficient method of cryopreservation of *Quercus suber* somatic embryos (Fernandes et al., 2008).

#### **2.2** *Quercus suber* **cryopreservation**

Propagation of cork oak presents several drawbacks as it has a high heterozigocity, often leading to individuals with high probability of instability and genetically distinct from parents, therefore leading to high numbers of undesired genotypes (Lopes et al., 2006). Moreover, seeds are only stored for short periods as they rapidly loose viability. This recalcitrance jeopardises the development of conservation and improvement programs in this species. As in most forest species, *Quercus suber* conservation approaches consist mainly in agro-forest sustainable systems, and scarce strategies using biotechnological approaches have already succeeded. Valladares et al. (2004) highlighted that highly interesting individuals may be maintained with vegetative propagation.

The cryopreservation of seeds or embryos seems therefore to have huge potential as an innovative preservation strategy, in particular in species with recalcitrance. González-Benito et al. (2002) examined different factors included in the cryopreservation protocols for *Quercus ilex* and *Q. suber* embryonic axes. The authors demonstrated that temperature of in vitro incubation played an important role, mostly for *Q. ilex* axes. *Q. suber* axes were sensitive to desiccation and cooling.

With respect to *Quercus* sp. somatic embryos, Martinez et al. (2003) and Valladares et al. (2004) successfully cryopreserved embryogenic cultures of *Q. robur* and *Q. suber*, using the vitrification method. As reported above, highly toxic cryoprotectants are used in most classical vitrification processes. To overcome these negative effects, recently our group (Fernandes et al., 2008) used a less toxic variation to the classical vitrification technique, called the encapsulation-dehydration method, to cryopreserve *Q. suber* material.

Nonetheless, during this process, usually complex and toxic solutions with high osmotic potential, such as the PVS2, are used for cryoprotection (Fernandes et al., 2008). Also the duration of the successive steps of a vitrification protocol is in general very short,

Some alternatives to vitrification-based techniques were developed, namely encapsulationdehydration strategies (e.g., Engelmann et al., 2008). Encapsulation-dehydration is based on concepts related with artificial seeds. Concisely, plant tissues, such as shoot tips or somatic embryos, are covered by for example alginate. Then, they are dehydrated (using exposures to highly concentrated solutions, and/or to air in a flow chamber), before being transferred to N2. This strategy has the advantage of using less toxic compounds such as glycerol than in other vitrification methods, thus minimizing stress conditions (e.g. Volk et al., 2006). It also has the advantage of easy and inexpensive manipulation, not requiring expensive

This method has been applied to many species, such as, for example, mulberry (Niino & Sakai, 1992), *Prunus* sp. (Brison et al., 1992), sweet potato (Feng et al., 2011; Hirai & Sakai, 2003), persimmon (Matsumoto et al., 2001), apple (Niino & Sakai, 1992; Paul et al., 2000), lily (Bouman & Klerk, 1990) and even grapevine embryogenic cell suspensions (Wang & Perl, 2006) or pear (Niino & Sakai, 1992; Scottez et al., 1992). In several assays as in those with *Robinia pseudoacacia*, it was demonstrated that encapsulation-dehydration originated better results than vitrification (Verleysen et al., 2005). Recently we have also demonstrated that encapsulation-dehydration was the most efficient method of cryopreservation of *Quercus* 

Propagation of cork oak presents several drawbacks as it has a high heterozigocity, often leading to individuals with high probability of instability and genetically distinct from parents, therefore leading to high numbers of undesired genotypes (Lopes et al., 2006). Moreover, seeds are only stored for short periods as they rapidly loose viability. This recalcitrance jeopardises the development of conservation and improvement programs in this species. As in most forest species, *Quercus suber* conservation approaches consist mainly in agro-forest sustainable systems, and scarce strategies using biotechnological approaches have already succeeded. Valladares et al. (2004) highlighted that highly interesting

The cryopreservation of seeds or embryos seems therefore to have huge potential as an innovative preservation strategy, in particular in species with recalcitrance. González-Benito et al. (2002) examined different factors included in the cryopreservation protocols for *Quercus ilex* and *Q. suber* embryonic axes. The authors demonstrated that temperature of in vitro incubation played an important role, mostly for *Q. ilex* axes. *Q. suber* axes were

With respect to *Quercus* sp. somatic embryos, Martinez et al. (2003) and Valladares et al. (2004) successfully cryopreserved embryogenic cultures of *Q. robur* and *Q. suber*, using the vitrification method. As reported above, highly toxic cryoprotectants are used in most classical vitrification processes. To overcome these negative effects, recently our group (Fernandes et al., 2008) used a less toxic variation to the classical vitrification technique,

called the encapsulation-dehydration method, to cryopreserve *Q. suber* material.

hampering the simultaneous treatment of a large number of samples.

instruments, as occurs in controlled freezing (Fernandes et al., 2008).

*suber* somatic embryos (Fernandes et al., 2008).

individuals may be maintained with vegetative propagation.

**2.2** *Quercus suber* **cryopreservation** 

sensitive to desiccation and cooling.

Fig. 4. a) Percentage of survival of cork oak cells after 10 weeks (for two protocols of cryopreservation used CRY25 and CRY35, corresponding to the final % content of water). b) Acclimatized plant obtained from cryopreserved somatic embryos that were recovered and cultivated on MSWH for maturation and conversion (adapted from Fernandes et al., 2008).

In this standard protocol for cork oak, somatic embryos or embryogenic clusters derived from mature trees and previously maintained on MS medium without growth regulators (MSWH) are used as samples. For encapsulation, embryos/clusters were separated and loaded in the alginate plus CaCl2 solutions, forming the beads (3–4 mm), each one contained one embryo/cluster. They were then pre-cultivated on sucrose-enriched standard liquid medium (MSWH with 0.7M sucrose) for 3 days. Beads were then desiccated by drying in the airflow of a laminar flow cabinet, and carefully weight loss was monitored for water content calculation. Two final water content (WC) values were assessed: 25% (CRY25) and 35% (CRY35). Afterwards, beads were placed in cryotubes (10 per vial), and immersed in N2 for 24 h. Samples' thawing was done by incubating the cvryotubes at 38 ºC (2 min) and incubating in detoxification solution (1 h). Beads were transferred to solid standard medium (MSWH) for regeneration (Figure 4a, b; Fernandes et al., 2008).

This cryopreservation technique developed by Fernandes et al. (2008) for cork oak somatic embryos is simple, effective and non-toxic for the species. Also, survival rates in encapsulated-dehydrated (but non-frozen) cork oak samples achieved 90%. We also demonstrated that cryopreserved somatic embryo derived clones were able to recovery, leading to plants morphologically normal and that had genetic stability.
