**1.2** *Quercus suber* **in vitro cloning: A reliable protocol?**

Cork oak (*Quercus suber* L.) belongs to Fagaceae, an important family of forest trees in the Northern hemisphere dominating temperate forests and Mediterranean ecosystems. In particular, cork oak is an abundant species in the Atlantic and West Mediterranean countries where it is an important component of Mediterranean ecosystems (Pinto et al., 2002). Cork is the bark of the oak, which is a natural, renewable and sustainable raw material product of economic interest for a range of applications. Due to its enormous economical importance, intense research has been focused on cork valuable material and, more recently, on cork oak germplasm-conservation programs (Fernandes et al., 2008).

In the last decades, studies were done to improve protocols of cork oak in vitro micropropagation and conservation (namely cryopreservation). In particular, the currently available rates of success in cork oak plant regeneration by somatic embryogenesis (Fernandes et al., 2011; Lopes et al., 2006; Loureiro et al., 2005; Pinto et al., 2001; 2002) and in cork oak material cryopreservation (Fernandes et al., 2008; Fernandes, 2011) are highly encouraging for researchers and breeders to consider the integration of these strategies in breeding and conservation programs of this species (Figure 1).

Recently, it was also established a Portuguese consortium to identify and characterize cork oak ESTs Gene responses to several biotic and abiotic stresses as well as to developmental conditioning are currently being screened and data will be of upmost importance to cork oak researchers and breeders (http://www.fct.pt/apoios/projectos/consulta/ projectos.phtml.en).

For long it has been assumed that *Quercus* species have, at some extent, recalcitrant responses to micropropagation in general, and to somatic embryogenesis in particular. Most common strategies for cork oak micropropagation use stem cuttings or use juvenile material or leaves for somatic embryogenesis (Santos, 2008; Fernandes et al., 2008). When utilizing material selected from adult field trees as explant sources, the use of greenhouse forced sprouts instead of directly collected field material is strongly advised.

The developed micropropagation by stem cutting is efficient with juvenile and, less, mature genotypes. Briefly, after disinfection with sodium hypochloride, explants (with 1-2 apical and/or lateral buds) are inoculated on WPM ("Woody Plant Medium", Lloyd and McCown, 1980) medium containing benzylaminopurine (BAP 0.5 mg/L) and naphthalene acetic acid (NAA 0.1 mg/L). After multiplication and elongation, shoots are exposed to an indol-butiric acid shock for rooting. Plants in this stage are then ready for acclimatization (Pires et al., 2003; Figure 2a).

conservation. The combination of these strategies will then enable large-scale production and disposition of tested clonal lines in industrial forest management. For example, micropropagation processes are well refined for spruce and larch species to support their commercial application (http://cfs.nrcan.gc.ca/ factsheets/conifersomatic). However, for most pine species, it is much more difficult to obtain somatic embryogenesis, though interesting advances are in course (e.g. Park et al., 2010). Similarly, the application of this technology to most forest dicotyledonous species, as is the case of *Quercus* genus, has demonstrated to be difficult due to species general recalcitrance to in vitro culture (Santos,

Cork oak (*Quercus suber* L.) belongs to Fagaceae, an important family of forest trees in the Northern hemisphere dominating temperate forests and Mediterranean ecosystems. In particular, cork oak is an abundant species in the Atlantic and West Mediterranean countries where it is an important component of Mediterranean ecosystems (Pinto et al., 2002). Cork is the bark of the oak, which is a natural, renewable and sustainable raw material product of economic interest for a range of applications. Due to its enormous economical importance, intense research has been focused on cork valuable material and, more recently, on cork oak germplasm-conservation programs (Fernandes et al., 2008).

In the last decades, studies were done to improve protocols of cork oak in vitro micropropagation and conservation (namely cryopreservation). In particular, the currently available rates of success in cork oak plant regeneration by somatic embryogenesis (Fernandes et al., 2011; Lopes et al., 2006; Loureiro et al., 2005; Pinto et al., 2001; 2002) and in cork oak material cryopreservation (Fernandes et al., 2008; Fernandes, 2011) are highly encouraging for researchers and breeders to consider the integration of these strategies in

Recently, it was also established a Portuguese consortium to identify and characterize cork oak ESTs Gene responses to several biotic and abiotic stresses as well as to developmental conditioning are currently being screened and data will be of upmost importance to cork oak researchers and breeders (http://www.fct.pt/apoios/projectos/consulta/

For long it has been assumed that *Quercus* species have, at some extent, recalcitrant responses to micropropagation in general, and to somatic embryogenesis in particular. Most common strategies for cork oak micropropagation use stem cuttings or use juvenile material or leaves for somatic embryogenesis (Santos, 2008; Fernandes et al., 2008). When utilizing material selected from adult field trees as explant sources, the use of greenhouse forced

The developed micropropagation by stem cutting is efficient with juvenile and, less, mature genotypes. Briefly, after disinfection with sodium hypochloride, explants (with 1-2 apical and/or lateral buds) are inoculated on WPM ("Woody Plant Medium", Lloyd and McCown, 1980) medium containing benzylaminopurine (BAP 0.5 mg/L) and naphthalene acetic acid (NAA 0.1 mg/L). After multiplication and elongation, shoots are exposed to an indol-butiric acid shock for rooting. Plants in this stage are then ready for acclimatization (Pires et al.,

**1.2** *Quercus suber* **in vitro cloning: A reliable protocol?** 

breeding and conservation programs of this species (Figure 1).

sprouts instead of directly collected field material is strongly advised.

2008).

projectos.phtml.en).

2003; Figure 2a).

Fig. 1. Schematic representation of a proposed strategy of integrating cork oak micropropagation and cryopreservation technologies in Portuguese breeding programs of this species (adapted from Santos, 2008).

As reported above, somatic embryogenesis is a regeneration strategy with enormous potential for breeding programs. Somatic embryos were developed in *Q. canariensis* (Bueno et al., 1996; 2000), *Q. rubra* (Vengadesan & Pijut, 2009), *Q. serrata* (Ishii et al., 1999; Takur et al., 1999), *Q. robur* (Cuenca et al., 1998; Endemann et al., 2002; Wilhelm et al., 1999), *Q. acutissima* (e.g., Kim, 2000) and *Q. petrea* (Chalupa, 2005). However, not only most studies use juvenile sources of explants (e.g., zygotic embryos and seedlings), but also plant conversion frequencies are still low, supporting the recalcitrance of these species.

*Q. suber* somatic embryogenesis was obtained first from juvenile plants (e.g., Bueno et al., 1996; 2000; Pinto et al., 2001) and later from leaf explants of mature plants (e.g., Hernandez et al., 2003; Lopes et al., 2006; Pinto et al., 2002; Santos et al., 2007) (Figure 2b,c).

Fig. 2. Micropropagation from field mature cork oak trees: a) Acclimatized plants micropropagated by stem cuttings; b) Scanning electon microscopy of two cotyledonary somatic embryos; c) converted embling (Adapted from Pires et al., 2003; Pinto et al., 2002; Santos, 2008).

Somatic Embryogenesis and Cryopreservation in Forest Species: The Cork Oak Case Study 401

The initially protocol developed by Santos and collaborators (Pinto et al., 2002; Lopes et al., 2006) was, however, not sufficiently efficient for large scale propagation and for immediate transfer to industrial breeding programmes of cork oak. Meanwhile, those and other authors reported the deficient maturation of somatic embryos during somatic embryogenesis, as the main cause for low conversion rates in this species (Chalupa, 2005; Fernandes et al 2011;

Hernández et al. (2003) highlighted that an adequate reserve deposition in the embryo tissues seems to be necessary for their adequate maturation. Efforts were made since then to manipulate physical conditions in order to promote the adequate accumulation of reserves (Fernandes, 2011; Santos, 2008). Fernández-Guijarro et al. (1995) reported that somatic embryos from cork oak young seedlings increased maturation under light followed by

Santos (2008) and Fernandes (2011) compared the accumulation profiles of carbohydrate, lipid and protein reserves during the maturation of cork oak somatic embryos and the zygotic counterparts. Assuming that the accumulation of reserves that occurs in zygotic embryos may be ideal for embryos maturation, Fernandes (2011) also compared the accumulation profiles of somatic embryos exposed to different conditions such as polyethylene glycol (PEG), abscisic acid (ABA) and cold, and defined the condition that led

From those analyses, the authors proposed an improvement to the initial somatic embryogenesis protocol developed by Pinto et al. (2002) (see Figure 3). In the improved protocol clusters of somatic globular embryos are isolated and transferred to MS medium (Murashige & Skoog, 1962) with PEG. After maturation, cotyledonar embryos are transferred to MS medium and submitted to chilling (4º C). Conversion is then achieved on woody plant medium (WPM) medium supplemented with BAP 0.5 mg/L and NAA 0.1

In conclusion, the inclusion of cold and osmotic stress in the protocol improved somatic embryos maturation and consequent conversion in approximately 70% of the genotypes. However, it was evident a genotype dependence in this process, with responsiveness ranging from very-good/in most genotypes to null, in few genotypes (Fernandes, 2011) (Figure 3).

The loss of embryogenic competence is one of the major drawbacks of long term micropropagation potocols (e.g., Brito et al., 2009). In particular, embryogenic masses were maintained for long periods may dedifferentiate and lose their embryogenic potential. In cork oak this phenomena has originated two types of calluses under the same conditions: embryogenic (EC) and non-embryogenic (NEC) and these last calluses rarely regain

In vitro functional changes during embryogenesis imply changes in explant cells from differentiated and quiescent (G0) stage to dedifferentiated dividing (G1-S-G2/M) stages, and later an evolution to embryogenic states. All these transitions imply changes in gene expression, and in cell cycle dynamics, where growth regulators, namely auxins and

Using embryogenic and non embryogenic calluses of adult cork oak genotypes, our group reported differential distribution of cells staged in G1, S and G2 phases according to callus

mg/L. After some weeks plants are acclimatized with success (Fernandes, 2011).

storage at 4 °C, and that controlled starvation could benefit synchronization.

to an accumulation profile closer to the one of the zygotic embryos.

**1.3 Current challenges for the SE process** 

cytokinins, are crucial players (Gahan, 2007).

embryogenic ability (Santos, 2008).

Hernández et al., 1999).

Fig. 3. Enhanced protocol of our group for cork oak somatic embryogenesis. MS medium - Murashige and Skoog, 1962; MSWH - MS medium with no growth regulators. (Adapted from Fernandes, 2011).

Fig. 3. Enhanced protocol of our group for cork oak somatic embryogenesis. MS medium - Murashige and Skoog, 1962; MSWH - MS medium with no growth regulators. (Adapted

from Fernandes, 2011).

The initially protocol developed by Santos and collaborators (Pinto et al., 2002; Lopes et al., 2006) was, however, not sufficiently efficient for large scale propagation and for immediate transfer to industrial breeding programmes of cork oak. Meanwhile, those and other authors reported the deficient maturation of somatic embryos during somatic embryogenesis, as the main cause for low conversion rates in this species (Chalupa, 2005; Fernandes et al 2011; Hernández et al., 1999).

Hernández et al. (2003) highlighted that an adequate reserve deposition in the embryo tissues seems to be necessary for their adequate maturation. Efforts were made since then to manipulate physical conditions in order to promote the adequate accumulation of reserves (Fernandes, 2011; Santos, 2008). Fernández-Guijarro et al. (1995) reported that somatic embryos from cork oak young seedlings increased maturation under light followed by storage at 4 °C, and that controlled starvation could benefit synchronization.

Santos (2008) and Fernandes (2011) compared the accumulation profiles of carbohydrate, lipid and protein reserves during the maturation of cork oak somatic embryos and the zygotic counterparts. Assuming that the accumulation of reserves that occurs in zygotic embryos may be ideal for embryos maturation, Fernandes (2011) also compared the accumulation profiles of somatic embryos exposed to different conditions such as polyethylene glycol (PEG), abscisic acid (ABA) and cold, and defined the condition that led to an accumulation profile closer to the one of the zygotic embryos.

From those analyses, the authors proposed an improvement to the initial somatic embryogenesis protocol developed by Pinto et al. (2002) (see Figure 3). In the improved protocol clusters of somatic globular embryos are isolated and transferred to MS medium (Murashige & Skoog, 1962) with PEG. After maturation, cotyledonar embryos are transferred to MS medium and submitted to chilling (4º C). Conversion is then achieved on woody plant medium (WPM) medium supplemented with BAP 0.5 mg/L and NAA 0.1 mg/L. After some weeks plants are acclimatized with success (Fernandes, 2011).

In conclusion, the inclusion of cold and osmotic stress in the protocol improved somatic embryos maturation and consequent conversion in approximately 70% of the genotypes. However, it was evident a genotype dependence in this process, with responsiveness ranging from very-good/in most genotypes to null, in few genotypes (Fernandes, 2011) (Figure 3).

#### **1.3 Current challenges for the SE process**

The loss of embryogenic competence is one of the major drawbacks of long term micropropagation potocols (e.g., Brito et al., 2009). In particular, embryogenic masses were maintained for long periods may dedifferentiate and lose their embryogenic potential. In cork oak this phenomena has originated two types of calluses under the same conditions: embryogenic (EC) and non-embryogenic (NEC) and these last calluses rarely regain embryogenic ability (Santos, 2008).

In vitro functional changes during embryogenesis imply changes in explant cells from differentiated and quiescent (G0) stage to dedifferentiated dividing (G1-S-G2/M) stages, and later an evolution to embryogenic states. All these transitions imply changes in gene expression, and in cell cycle dynamics, where growth regulators, namely auxins and cytokinins, are crucial players (Gahan, 2007).

Using embryogenic and non embryogenic calluses of adult cork oak genotypes, our group reported differential distribution of cells staged in G1, S and G2 phases according to callus

Somatic Embryogenesis and Cryopreservation in Forest Species: The Cork Oak Case Study 403

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

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

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

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

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).

may arise after long term culture (Brito et al., 2009).

cultures, embryos and seeds (Feng et al., 2011).

and survival rates of cells.

inside the cell (e.g., Sakai et al., 2008).

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 (Fernandes, 2011).

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; for review see Dewitte & Murray, 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, 2003; Gahan et al., 2007).

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