**1. Introduction**

396 Current Frontiers in Cryopreservation

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#### **1.1 General concepts**

It is widely accepted that propagation and conservation strategies are crucial in any forest breeding program and that plant biotechnology has powerful tools to propagate and preserve selected genotypes (Jain, 1999; Park et al., 1988; Park, 2002). In particular, in vitro techniques have demonstrated to be essential in a large number of agricultural and forest breeding programs, allowing large scale clonal propagation of elite genotypes (Pinto et al., 2008) or of endangered forest species (Brito et al., 2009). In vitro techniques are also essential in breeding programs involving genetic improvement (e.g. using techniques such as somatic hybridization or genetic transformation) or involving long term germplasm conservation by cryopreservation (Benson, 2008; Fernandes et al., 2008).

Somatic embryogenesis offers advantages in improving forest species over other in vitro propagation methods (Park, 2002), namely: a) somatic embryos simultaneously possess both the shoot and root meristems; so a distinct rooting stage, which usually involves stressing procedures, is not necessary; b) during somatic embryogenesis, embryos/clusters are often formed faster and potentially at extremely high numbers per explant; c) somatic embryogenesis is amenable to automation, which means it may become cheaper than other clonal propagation techniques (Pinto et al., 2002; 2008); d) finally, a robust and efficient protocol of somatic embryogenesis will allow that embryogenic clonal lines can be preserved for long periods (e.g., in liquid N2) while corresponding plants are transferred to field conditions and are monitored for their characteristics.

The combination of somatic embryogenesis and conservation strategies in breeding programs allows that one may develop high-value forest clonal varieties merely by recovering from N2 those genotypes/clones that showed best characteristics in the field. These selected genotypes can later be used for advanced breeding programs and commercial forestry (Park, 2002).

From the exposed advantages, it is generally accepted among breeders that breeding programs using in vitro clonal strategy require as a pre-requisite that a cloning technique (preferably somatic embryogenesis) is well established. Then it also requires the optimization of long-term genetic testing methodologies of clonal lines and long term

Somatic Embryogenesis and Cryopreservation in Forest Species: The Cork Oak Case Study 399

Fig. 1. Schematic representation of a proposed strategy of integrating cork oak

conversion frequencies are still low, supporting the recalcitrance of these species.

et al., 2003; Lopes et al., 2006; Pinto et al., 2002; Santos et al., 2007) (Figure 2b,c).

this species (adapted from Santos, 2008).

Santos, 2008).

micropropagation and cryopreservation technologies in Portuguese breeding programs of

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

*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

a b 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;

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, 2008).
