Cryopreservation of Quercus suber somatic embryos - Tree Physiology

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
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