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

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,

Somatic Embryogenesis and Cryopreservation in Forest Species: The Cork Oak Case Study 407

fidelity, for the parameters used: in all samples, both ploidy and DNA content were in concordance with literature data: 2C= 1.90 pg DNA (Loureiro et al., 2005; Santos et al., 2007), and changes in DNA content of non-cryopreserved and cryopreserved samples were

Flow cytometric techniques, despite highly rapid and robust, may however not detect minor changes in DNA content, so it must be emphasised that the putative occurrence of small

a b c Fig. 5. Flow cytometric estimation of genome size of *Q. suber* samples of control (a) and after cryopreservation: CRY25 (b) and CRY35 (c). In all graphics peaks 1 and 3 correspond to 2C and 4C nuclei of cork oak somatic embryos, while peaks 2 and 4 correspond to the internal soy standard (adapted from Fernandes et al., 2008). PI: intensity of propidium fluorescence.

**Tissue DI SD 2c DNA (pg) SD 1c DNA** 

**Fresh** 0.774a 0.0059 1.93 0.015 946 **25% Cryc** 0.777a 0.0073 1.94 0.018 950 **35% Cryc** 0.772a 0.0138 1.93 0.034 944

Table 1. Nuclear DNA content of *Quercus suber* L. fresh and cryopreserved embryos. The results are given as mean and standard deviation (SD) of the 2C DNA content in mass values (pg). Nuclear DNA content in Mbp is also given. a Mean values followed by the same letter are not significantly different according to the Tukey-Kramer multiple comparison test at P 0.01. Note: b1 pg DNA = 978 Mbp. cCryopreserved embryos (25% and 35 % water

Both works supported therefore that the protocols for somatic embryogenesis and cryopreservation developed so far for cork oak led to "true-to-typeness" (for ploidy and

As explored above, molecular markers provide information on sequence mutation. This information is therefore complementary to the information provided by flow cytometry (or

DNA amount parameters) and were worthy of use in cork oak breeding programs.

**(Mbp)b** 

changes in DNA content in these kinds of assays should not be discarded.

minimal (< 0.01 pg/2C) (Figure 6; Table 1).

content) (adapted from Fernandes et al 2008).

even by chromosome counting).

any protocol of plant cloning or germplasm conservation should lead to no somaclonal variation.

Genetic variability of in vitro material or of cryopreserved samples may be assessed by several techniques. Molecular and genetic techniques when used individually give a limited perspective of the occurrence of somaclonal variation, but if combined may provide an interesting and complementary toolbox of markers for "true-to-typeness" evaluation (e.g., Santos et al., 2007).

Fig. 5. Flow cytometry histograms of relative propidium iodide (PI) fluorescence intensity obtained after simultaneous analysis of nuclei isolated from *Quercus suber* and from the reference standard *Glycine max* (2C=2.50 pg DNA): **a)** leaves of the mother plant; **b)** somatic embryo. Histogram *peak 1:* nuclei at G0/G1 phase of *Q. suber*; *peak 2:* nuclei at G0/G1 phase of *G. max*, *peak 3* nuclei at G2 phase of *Q. suber*, *peak 4* nuclei at G2 phase of *G. max* (adapted from Loureiro et al., 2005).

Gross genetic variations of genomic origin affect mostly the number of chromosomes and ploidy level and can be detected by chromosome counting or flow cytometry (Loureiro et al., 2005). Chromosome mutations (e.g., inversion, translocation) and genic mutations are screened by molecular markers that detect DNA sequence modifications. Among these, the most popular are RFLPs (restriction fragment length polymorphisms), RAPDs (randomly amplified polymorphic DNAs), AFLPs (amplified fragment length polymorphisms) or microsatellites/SSRs (simple sequence repeats). RFLPs and AFLPs are highly reproducible techniques but are costly and may be time-consuming. For easy application of SSRs, it is required that the microsatellite loci and its flanking primers are readily available for a given species, while RAPDs is simple but may have a lack of reproducibility.

Several genetic and molecular markers have been used to assess somaclonal variation in micropropagated oak plants. Loureiro et al. (2005), using flow cytometry, found no ploidy or DNA content variations in cork oak embryogenic tissues or among somatic embryos (Figure 5).

Also, Fernandes et al. (2008) using cryopreserved material, confirmed by flow cytometric analyses that the two cryopreservation procedures (CRY25 and CRY35) provided genetic

any protocol of plant cloning or germplasm conservation should lead to no somaclonal

Genetic variability of in vitro material or of cryopreserved samples may be assessed by several techniques. Molecular and genetic techniques when used individually give a limited perspective of the occurrence of somaclonal variation, but if combined may provide an interesting and complementary toolbox of markers for "true-to-typeness" evaluation (e.g.,

a b Fig. 5. Flow cytometry histograms of relative propidium iodide (PI) fluorescence intensity obtained after simultaneous analysis of nuclei isolated from *Quercus suber* and from the reference standard *Glycine max* (2C=2.50 pg DNA): **a)** leaves of the mother plant; **b)** somatic embryo. Histogram *peak 1:* nuclei at G0/G1 phase of *Q. suber*; *peak 2:* nuclei at G0/G1 phase of *G. max*, *peak 3* nuclei at G2 phase of *Q. suber*, *peak 4* nuclei at G2 phase of *G. max* (adapted

Gross genetic variations of genomic origin affect mostly the number of chromosomes and ploidy level and can be detected by chromosome counting or flow cytometry (Loureiro et al., 2005). Chromosome mutations (e.g., inversion, translocation) and genic mutations are screened by molecular markers that detect DNA sequence modifications. Among these, the most popular are RFLPs (restriction fragment length polymorphisms), RAPDs (randomly amplified polymorphic DNAs), AFLPs (amplified fragment length polymorphisms) or microsatellites/SSRs (simple sequence repeats). RFLPs and AFLPs are highly reproducible techniques but are costly and may be time-consuming. For easy application of SSRs, it is required that the microsatellite loci and its flanking primers are readily available for a given

Several genetic and molecular markers have been used to assess somaclonal variation in micropropagated oak plants. Loureiro et al. (2005), using flow cytometry, found no ploidy or DNA content variations in cork oak embryogenic tissues or among somatic embryos

Also, Fernandes et al. (2008) using cryopreserved material, confirmed by flow cytometric analyses that the two cryopreservation procedures (CRY25 and CRY35) provided genetic

species, while RAPDs is simple but may have a lack of reproducibility.

variation.

Santos et al., 2007).

from Loureiro et al., 2005).

(Figure 5).

fidelity, for the parameters used: in all samples, both ploidy and DNA content were in concordance with literature data: 2C= 1.90 pg DNA (Loureiro et al., 2005; Santos et al., 2007), and changes in DNA content of non-cryopreserved and cryopreserved samples were minimal (< 0.01 pg/2C) (Figure 6; Table 1).

Flow cytometric techniques, despite highly rapid and robust, may however not detect minor changes in DNA content, so it must be emphasised that the putative occurrence of small changes in DNA content in these kinds of assays should not be discarded.

Fig. 5. Flow cytometric estimation of genome size of *Q. suber* samples of control (a) and after cryopreservation: CRY25 (b) and CRY35 (c). In all graphics peaks 1 and 3 correspond to 2C and 4C nuclei of cork oak somatic embryos, while peaks 2 and 4 correspond to the internal soy standard (adapted from Fernandes et al., 2008). PI: intensity of propidium fluorescence.


Table 1. Nuclear DNA content of *Quercus suber* L. fresh and cryopreserved embryos. The results are given as mean and standard deviation (SD) of the 2C DNA content in mass values (pg). Nuclear DNA content in Mbp is also given. a Mean values followed by the same letter are not significantly different according to the Tukey-Kramer multiple comparison test at P 0.01. Note: b1 pg DNA = 978 Mbp. cCryopreserved embryos (25% and 35 % water content) (adapted from Fernandes et al 2008).

Both works supported therefore that the protocols for somatic embryogenesis and cryopreservation developed so far for cork oak led to "true-to-typeness" (for ploidy and DNA amount parameters) and were worthy of use in cork oak breeding programs.

As explored above, molecular markers provide information on sequence mutation. This information is therefore complementary to the information provided by flow cytometry (or even by chromosome counting).

Somatic Embryogenesis and Cryopreservation in Forest Species: The Cork Oak Case Study 409

Table 2. Characteristics of the microsatellite loci amplified in *Q. suber*. Allele size found in this study and allele size range and number of alleles (in parenthesis) found in other publications are also given: values for QrZAG7 and QrZAG11 are from Hornero et al.

Finally, Wilhelm et al. (2005) found that that during somatic embryogenesis process of *Q. robur*, genetic instability occurred. Also, Lopes et al. (2006) confirmed genetic stability of somatic embryos and emblings derived from our somatic embryogenesis protocol (Lopes et al., 2006; Pinto et al., 2002) and after, Fernandes et al. (2008) also confirmed this stability using SSRs in material after recovering from cryopreservation (Table 2). Together with this overall genetic stability, the regenerated cork oak plants looked normal, healthy and well developed shoot. The authors concluded that the encapsulation-dehydration cryopreservation protocol used in cork oak somatic embryos was an efficient method of storage, regarding several parameters: recovery and survival rates and genetic/morphologic stability. To support the battery of protocols for molecular and genetic analyses of cork oak, Santos et al. (2007) published in detail the reliable protocol for analysis of cork oak material by SSRs and ploidy/nDNA quantification, where technical aspects and potential troubleshooting that may occur during analysis of this material are deeply discussed.

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

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.

 QsG0 QsG5 QsGM1 QsGM2 QM58TGT 201 185/211 203/210 203/210 QM50-3M 276/286 285/287/292 278 282/288 QpZAG9 223/238 223/233 218/223 223/233 QpZAG15 108/123 106/120 nd nd QpZAG36 209/219 216/220 207/209 207/209 QpZAG110 223/233 220/238 221 221/233 QrZAG7 106/119 115/127 106/121 106/121 QrZAG11 229/263 261/273 nd nd

Locus Allele size (bp)

(2001*b*), values for the 6 remaining loci are from Lopes et al. (2006).

**4. Concluding remarks** 

obtained.

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 al., 2011).

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

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



Table 2. Characteristics of the microsatellite loci amplified in *Q. suber*. Allele size found in this study and allele size range and number of alleles (in parenthesis) found in other publications are also given: values for QrZAG7 and QrZAG11 are from Hornero et al. (2001*b*), values for the 6 remaining loci are from Lopes et al. (2006).

Finally, Wilhelm et al. (2005) found that that during somatic embryogenesis process of *Q. robur*, genetic instability occurred. Also, Lopes et al. (2006) confirmed genetic stability of somatic embryos and emblings derived from our somatic embryogenesis protocol (Lopes et al., 2006; Pinto et al., 2002) and after, Fernandes et al. (2008) also confirmed this stability using SSRs in material after recovering from cryopreservation (Table 2). Together with this overall genetic stability, the regenerated cork oak plants looked normal, healthy and well developed shoot. The authors concluded that the encapsulation-dehydration cryopreservation protocol used in cork oak somatic embryos was an efficient method of storage, regarding several parameters: recovery and survival rates and genetic/morphologic stability. To support the battery of protocols for molecular and genetic analyses of cork oak, Santos et al. (2007) published in detail the reliable protocol for analysis of cork oak material by SSRs and ploidy/nDNA quantification, where technical aspects and potential troubleshooting that may occur during analysis of this material are deeply discussed.
