Cryopreservation of Quercus suber somatic embryos - Tree Physiology

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1846 FERNANDES ET AL. Figure 4. Flow cytometric estimation of nuclear genome size of Quercus suber somatic embryos. Histograms of relative fluorescence obtained after simultaneous analysis of nuclei isolated from Q. suber and the internal soy reference standard (Glycine max cv. Polanka). Peaks 1 and 3 correspond to G0/G1 and G2 nuclei of cork oak somatic embryos, respectively, and Peaks 2 and 4 correspond to the internal soy standard. Mean channel number, DNA index (DI = mean channel number of sample/mean channel number of reference standard) and coefficient of variation (CV, %) value of each peak are shown. Abbreviations: CRY25 and CRY35 denote cryopreserved embryos desiccated to 25 and 35% water content, respectively. TREE PHYSIOLOGY VOLUME 28, 2008 ing in size from 175 to 228 bp. Allele sizes were well within the range of published values. All loci were heterozygous and no differences in banding pattern were observed between the treatments and control (Figure 6). Discussion We developed a simple encapsulation and desiccation technique to prepare somatic embryos of cork oak for cryopreservation. Treated embryos displayed high viability and genetic stability. Successful cryopreservation of somatic embryos from adult cork oak could greatly facilitate the use of somatic embryogenesis in breeding programs with this species. Encapsulation and desiccation had no detectable adverse effects, as indicated by the 100% viability of non-frozen control samples. The method, unlike vitrification, does not require toxic cryoprotectants such as PVS2 (Volk et al. 2006), thus minimizing the risk of tissue injury. Similarly, with Robinia pseudoacacia somatic embryos, better survival was obtained by encapsulation and desiccation (87%) than by vitrification (78%) (Verleysen et al. 2005a). Cryopreservation following encapsulation and desiccation has also proved effective with somatic embryos of other woody species such as Citrus sp. (Gonzalez-Arnao et al. 2003) and Melia azedarach L. (Scocchi et al. 2007). Viability of Quercus suber embryos dried to either 25% or 35% WC was high. Water content is a key parameter in cryopreservation experiments, because plant cells can be damaged by severe dehydration or ice crystal formation during freezing (Engelmann 2000). The optimal WC is species-dependent and can be defined as the value that allows freezing with the least injury to cellular components as measured by survival after cryopreservation: e.g., 33% for apple (Niino and Sakai 1992), 38.6% for azalea (Verleysen et al. 2005b) and 19% for eucalypts (Pâques et al. 2002). Clonal forestry strategies based on cryopreservation and in vitro culture (e.g., somatic embryos) may be advantageous. For example, Takagi et al. (1997) verified that cryopreserved taro (Colocasia esculenta (L.) Schott) shoot tips developed without intermediate callus formation, thus minimizing the risk of somaclonal variation usu- Table 4. Nuclear DNA content of non-frozen (control) and cryopreserved (CRY25 and CRY35) encapsulated and desiccated embryos of Quercus suber. Values are means (± SD, n = 3) of the 2C DNA content in mass values (pg). Nuclear DNA content (Mbp) and mean coefficient of variation values (CV) obtained for the G0/G1 DNA peak are shown. Mean DI values were not significantly different according to the Tukey-Kramer multiple comparison test at P < 0.01. Abbreviations: CRY25 and CRY35 denote cryopreserved embryos desiccated to 25 and 35% water content, respectively. One pg DNA = 978 Mbp (Dolezel et al. 2003). Tissue DI 2C DNA (pg) 1C DNA (Mbp) CV (%) Control 0.774 ± 0.006 1.93 ± 0.02 946 2.94 CRY25 0.777 ± 0.007 1.94 ± 0.02 950 3.70 CRY35 0.772 ± 0.014 1.93 ± 0.03 944 3.13 Downloaded from http://treephys.oxfordjournals.org/ by guest on March 21, 2013
ally associated with callus formation. Cryopreservation may therefore prove generally effective in reducing intermediate callus formation during clonal propagation (Takagi 2000). It is critical that embryogenic clones are maintained indefinitely, without change in genetic makeup or loss of viability during cryogenic storage (Park 2002). Cryopreservation may induce genetic instability (Sakai 2004); however, the number of studies on the occurrence of genetic variation in cryopreserved samples remains limited (e.g., Panis and Lombardi 2005). For example, no genetic stability evaluations were performed in previously reported cryopreservation experiments with embryogenic cultures of Q. suber (Martinez et al. 2003, Valladares et al. 2004). However, Hao et al. (2002a) successfully cryopreserved cell suspensions of Citrus sp. without ploidy variation and randomly amplified polymorphic DNA (RAPD) variations. In addition, surviving cells of some genotypes regenerated somatic embryos with better performance than the controls. Cryopreserved strawberry shoot tips also showed ploidy stability, and AFLP analyses revealed only one band differing from non-cryopreserved samples (Hao et al. Figure 6. Amplification of MQS13 locus in control, CRY25 and CRY35 surviving explants of Quercus suber. The pattern shows heterozygous individuals with two alleles of 228 and 217 bp (left to right) in Lanes 1, 2 and 3. Standard fragment sizes in Lane 4 are 255, 204 and 200 bp (left to right). Abbreviations: CRY25 and CRY35 denote cryopreserved embryos desiccated to 25 and 35% water content, respectively. CRYOPRESERVATION OF CORK OAK SOMATIC EMBRYOS 1847 TREE PHYSIOLOGY ONLINE at http://heronpublishing.com Figure 5. Three AFLP polymorphisms between CRY25 and control somatic embryos of Quercus suber, confirmed in repeated reactions. (A and B) The two polymorphic bands generated with the primer combination E-AGC/M-CAA. Alleles were present only in the CRY25 sample, at 128 and 199 bp, respectively. (C) One polymorphic fragment (182 bp) between CRY25 and the control, generated with the primer combination E-ACG/M-CAC. Abbreviations: CRY25 and CRY35 denote cryopreserved embryos desiccated to 25 and 35% water content, respectively. 2002b). We assessed the genetic stability of recovered CRY25 and CRY35 material by a battery of techniques that gave complementary information on ploidy levels, nDNA content, AFLP and SSR. Flow cytometry confirmed the genetic stability of the cryopreserved material. Both ploidy level and DNA content were consistent with published data: 2C = 1.90 pg DNA (Loureiro et al. 2005, Santos et al. 2007). Moreover, variation in DNA content between control and cryopreserved samples was low (0.01 pg), as was variation within treatments. Although all CV values were within the acceptance criteria for FCM samples (i.e. below 5%), as stated by Galbraith et al. (2002), small changes in DNA content may have occurred. The typing of four SSR loci detected no genetic variability, supporting our FCM results. The genotyping accuracy of the selected markers was previously tested in Quercus spp. (Dow et al. 1995, Dow and Ashley 1996, Steinkellner et al. 1997). Samples were diploid and heterozygous as expected, and no polymorphisms were found between controls and treatments based on the resolution of this analysis with four microsatellite loci. Wilhelm et al. (2005) used two microsatellite loci, MSQ4 and MSQ13, to assess genetic instability during somatic embryogenesis in Q. robur and detected variation among somatic embryos within all embryogenic lines, though no genetic instability was found among the regenerated plants. Lopes et al. (2006), who used other microsatellite loci, reported the occurrence of one mutation, representing a low mutation rate of 2.5%. Genetic fingerprinting based on AFLP allowed direct analysis of variation at loci spread over the whole genome. No consistent polymorphisms were found between control and CRY35, indicating a perfect match between the control and CRY35 AFLP-fingerprints. The presence of three extra AFLP-bands in CRY25, which were not detected in the control sample or in CRY35, might be the result of putative small mutations, DNA methylation or cryo-selection of subpopulations Downloaded from http://treephys.oxfordjournals.org/ by guest on March 21, 2013
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Cryopreservation of Quercus suber somatic embryos - Tree Physiology

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