**4. Concluding remarks**

408 Current Frontiers in Cryopreservation

Fig. 6. DNA profiles generated by the RAPD primers OPS 17, 18 and 19, in the three different stages of the somatic embryogenesis process: donor plant (DP), somatic embryo (SE) and embling (EM). M, size marker (1Kb Plus DNA Ladder) (adapted from Fernandes et

In micropropagated *Q. serrate* no aberrations in the banding patterns were detected by RAPD markers (Thakur et al., 1999). Also, RAPDS were used to assess putative occurrence somaclonal variation in *Q. suber* embryogenic lines, but no molecular changes were found (Gallego et al., 1997, Sanchez et al., 2003). More recently, RAPDS were also used to evaluate genetic instability of somatic embryos of *Q. suber* obtained by the above described protocols

Techniques for RAPD analyses however pose several problems of reproducibility, and give restricted information. So, other molecular analyses can provide complementary information to RAPDS. Microsatellites and AFLP are among the most used markers in *Quercus*. AFLP markers detected changes in cork oak embryogenic lines (Hornero et al., 2001). We also used AFLP to test putative genetic instability during the developed cryopreservation/somatic embryogenesis processes of cork oak (Fernandes et al., 2008). It was used six primer sets that revealed an overall high proximity value between the two vitrification-encapsulation cryopreserved (CRY25 and CRY35) and control samples. Occasionally, few extra AFLP-bands in CRY25 samples were detected and the occurrence of putative small mutations, or DNA methylation or even to subpopulation cryo-selection

al., 2011).

(Figure 6; Fernandes et al., 2011).

should not be excluded (Fernandes et al., 2008).

The utility of plant biotechnology tools in woody forest species propagation and preservations has been recognised decades ago, but only recently, it has been effectively incorporated in industrial breeding programs. Despite no robust and efficient protocol for cork oak regeneration by somatic embryogenesis is available yet, the advances observed in the last decade, together with the already available protocol for cryopreservation of this species, open perspectives for the incorporation of these two approaches in future breeding program of this species. Moreover, with the available protocols stable genotypes were obtained.

Genetic and molecular stability was assessed using complementary genetic and molecular techniques such as flow cytometry, RAPDS, AFLP and SSRs. Finally, an interesting research field will focus on the control of cell cycle progression order to control different stages of somatic embryogenesis and preservation. It is our believe that by manipulating proteins that control cell cycle phases transition (which are at the basis of differentiated/ undifferentiated cells) we´ll be able to manipulate the reversion phenomena between NEC and EC and also to better control the developmental somatic embryogenesis stages.

Somatic Embryogenesis and Cryopreservation in Forest Species: The Cork Oak Case Study 411

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Fernandes P, E Rodriguez, G Pinto, I Roldán-Ruiz, M DeLoose & C Santos (2008)

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**Proline and the Cryopreservation of** 

Cryopreservation has been proven to be an effective technology for the cost-effective, longterm preservation of genetic material. A wide range of plant material including cultured cells, tissues, embryos, meristems, pollen and seeds can be effectively preserved for extended periods of time and, when thawed, can be used to rapidly produce stock plants, with good preservation of genetic and physiological characteristics. Numerous protocols including controlled rate cooling, PVS2 vitrification, encapsulation-vitrification, and encapsulation-dehydration have been developed that allow the cryopreservation of a wide range of plant germplasm (Burritt, 2008), but irrespective of the protocol used each step in a cryopreservation protocol has the potential to impose a stress on plant cells. Low temperatures that lead to freezing can impose stress on cells and tissues in two ways, by the direct effects of low temperatures on cell function and integrity or by the cellular dehydration that occurs when the cells water freezes. Several of the mechanisms by which

Numerous studies have shown that cold temperatures induce the accumulation of metabolites, including low-molecular-weight carbohydrates such as fructose, glucose, maltose and raffinose, and amino acids such as proline and glutamine (Taji et al., 2002; Cook et al., 2004). These metabolites play important protective roles in freezing tolerance in whole plants (Kaplan and Guy, 2004) and this has lead to their extensive use in the protocols developed for the cryopreservation of isolated plant cells and tissues (Burritt, 2008). In particular, the amino acid proline has been found to help confer freezing tolerance in a wide variety of both animal and plant cells, and is often added to cryoprotective solutions or is used for preconditioning plants or pretreating isolated cells or tissues prior to cryopreservation (Burritt, 2008). Despite its widespread use, little is known of the

This chapter gives an overview of proline synthesis and metabolic regulation in plants and the changes in proline metabolism associated with desiccation and freezing tolerance, which are both of importance for the successful cryopreservation of plants cells and tissues. The

these two forms of stress can damage plant cells are shown in Figure 1.

mechanisms via which proline protects cells during cryopreservation.

**1. Introduction** 

**Plant Tissues: Functions and** 

**Practical Applications** 

David J. Burritt

*New Zealand* 

*The Department of Botany, The University of Otago,Dunedin* 

Wilhelm E, K Hristoforoglu, S Fluch & K Burg (2005) Detection of microsatellite instability during somatic embryogenesis of oak. Plant Cell Reports, 23: 790-795 **20**
