![Cooling time courses of fully hydrated (A, B) and dried (C, D) embryonic axes of Quercus embryos. In (A), bare embryos of Q. franchetii, Q. schottkyana, Q. gambelii (WY) and Q. rubra (see key) were plunged into N 2 slush and warmed in 40 8C water [only Q. gambelii (WY) is shown]. Panel (B) compares cooling time courses of embryos of Q. franchetii cooled using various methods as indicated. Panels (C) and (D) give representative time courses for Q. rubra embryos dried for 0, 45 and 120 min and then cooled in N 2 slush or in cryovials placed in vapour above liquid nitrogen-LN. The encircled discontinuities indicate ice formation. Sample sizes and statistics are summarized in Table 6.](https://www.researchgate.net/profile/Ke-Xia-4/publication/267044836/figure/fig1/AS:614183833976839@1523444245058/Cooling-time-courses-of-fully-hydrated-A-B-and-dried-C-D-embryonic-axes-of-Quercus_Q320.jpg)
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Factors affecting stress tolerance in recalcitrant embryonic axes from seeds
of four Quercus (Fagaceae) species native to the USA or China
Ke Xia1,2, Lisa M. Hill2, De-Zhu Li1,3 and Christina Walters2,*
1
Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China,
2
USDA-ARS National Center for Genetic Resources Preservation, Fort Collins, CO 80524, USA and
3
Key Laboratory of
Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201, China
*For correspondence. E-mail christina.walters@ars.usda.gov
Received: 15 May 2014 Returned for revision: 11 June 2014 Accepted: 20 August 2014
†Background and Aims Quercusspecies are often considered ‘foundation’ components of several temperateand/or
subtropical forest ecosystems. However, the populations of some species are declining and there is considerable
urgency to develop ex situ conservation strategies. In this study, the storage physiology of seeds within Quercus
was explored in order to determine factors that affect survival during cryopreservation and to provide a quantitative
assessment of seed recalcitrance to support future studies of this complex trait.
†Methods Water relations and survival of excised axes in response to water loss and cryo-exposure were compared for
four Quercu s speciesfrom subtropical China (Q. franchetii,Q. schottkyana)andtemperateUSA(Q. gambelii,Q. rubra).
†Key Results Seed tissues initially had high water contents and water potentials. Desiccation tolerance of the em-
bryonic axis was not correlatedwith the post-shedding rainfall patterns where the samples originated. Instead, higher
desiccation tolerance was observed in samples growing in areas with colder winters. Survival following cryo-expos-
ure correlated with desiccation tolerance. Among species, plumule tissues were more sensitive than radicles to exci-
sion, desiccation and cryo-exposure, and this led to a higher proportion of abnormally developing embryos during
recovery following stress.
†Conclusions Quercus species adapted to arid and semi-humid climates still produce recalcitrant seeds. The ability
to avoid freezing rather than drought may be a more important selection factor to increase desiccation tolerance.
Cryopreservation of recalcitrant germplasm from temperate species is currently feasible, whilst additional protective
treatments are needed for ex situ conservation of Quercus from tropical and subtropical areas.
Key words: Seed desiccation tolerance, recalcitrance, embryonic axis, critical moisture content, cryopreservation, oak,
Cyclobalanopsis, subtropical, plumule, Quercus gambelii,Quercus rubra,Quercus franchetii,Quercus schottkyana.
INTRODUCTION
Seeds are broadly classified as ‘recalcitrant’ and ‘orthodox’
based on their tolerance of desiccation. Desiccation tolerance
has often been considered a qualitative feature of the species,
with seeds either perishing or surviving after some level of
drying (for example to ambient relative humidity). However,
we know that recalcitrant seeds exhibit a range of tolerances to
desiccation (Walters and Koster, 2007;Berjak and Pammenter,
2008) and that the extent of tolerated water loss depends on the
intensity and duration of drying, the method and tissues used to
assess survival, seed source, handling procedures and a host of
other factors. The complexity of the incidence and measurement
of seed recalcitrance impedes our abilities to probe it and to ul-
timately understand the mechanisms of damage or protection
that contribute to variation in post-harvest physiology among
all seeds. Comparative studies of the genetic regulation and eco-
logical significance of the complex trait of seed recalcitrance will
require reliable, quantitative assessments of the seed recalcitrance
phenotype. Future studies will be expedited by demonstrated and
repeatable phenotypic variation among related species or popula-
tions distributed across a range of climates.
Habitat or climate characteristics appear to contribute to vari-
ation in seed desiccation tolerance among populations. For
example, Coffea species adapted to longer dry seasons tended
to survive to lower water contents (Dussert et al., 2000;Eira
et al.,2006). Desiccation tolerance in seeds of Quercus petraea,
Aesculus hippocastanum,Acer pseudoplatanus,Zizania palustris
and Camellia sinensis was related to duration of the growing
season, which likely influences embryo maturity (Berjak et al.,
1993;Vertucci et al.,1994a;Daws et al., 2004,2006;Daws and
Jensen, 2011). Seeds of tropical rainforest species are reputed to
be highly recalcitrant, despite the long growing season, because
adaptations for seed desiccation tolerance were lost as species
evolved in climates without seasonal drought or freezing tempera-
tures (Farnsworth, 2000). Collectively, past research presents an
apparent paradox about the evolution of seed recalcitrance in
that recalcitrance maybe favouredin areas without winter freezing
temperatures as well asareas with short growing seasons bracketed
by freezing winter temperatures.
The genus Quercus (Fagaceae) may be an ideal study system to
explore the seed recalcitrance trait and to provide a set of carefully
described, closely related species with known variation in seed
physiology in anticipation of future genomic and ecological
studies. Over 450 species of Quercus trees or shrubs are broadly
distributed across the Northern Hemisphere in habitats ranging
from temperate and tropical forests to semi-deserts and arid envir-
onments (Nixon, 1993,1997;Huanget a l.,1999). Quercus species
Published by Oxford University Press on behalf of the Annals of Botany Company 2014.
This work is written by (a) US Government employee(s) and is in the public domain in the US.
Annals of Botany Page 1 of 13
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are often considered a ‘foundation’ component of the ecosystems
they inhabit (Ellison et al., 2005), yet population sizes are declin-
ing as a result of disease, habitat loss or climate change. Hence,
there is considerable urgency to develop ex situ conservation strat-
egies to preserve the genetic diversity extant in wild Quercus
populations (Kueppers et al., 2005;Tyler et al.,2006;Li and
Pritchard, 2009).
The known recalcitrant physiology of seeds from several
Quercus species complicates ex situ conservation efforts
(Pritchard, 1991;Pence, 1992;Gonza
´lez-Benito and Martı
´n,
2002;Gonza
´lez-Benito et al.,2002;Fernandes et al., 2008;
Chmielarz et al., 2011;Ganatsas and Tsakaldimi, 2013;Walters
et al.,2013). Because of their relatively high water content, recal-
citrant seeds are susceptible to lethal damage during conventional
freezer storage (Li and Pritchard, 2009;Walt ers et al., 2013). Many
efforts to cryopreserve recalcitrant seeds focus on rapid drying of
excised embryonic axes followed by rapid cooling (Wesley-Smith
et al., 1992,2001,2004;Walters et al., 2008). A target water
content of 0.25 g H
2
O
–1
(dry mass – lipid mass) is sufficiently
dry to reduce the probability of lethal ice formation in cells that
are cooled to liquid nitrogen temperatures at rates faster than
10 8Cmin
–1
(0.167 8Cs
–1
)(Vertucci, 1989;Pence, 1992;
Wesley-Smith et al., 1992,2001,2004,2014). For this reason, a
water content of 0.25 g H
2
Og
–1
is often considered a benchmark
for cryopreservability and water at lower water contents is often
described as unfreezable (Wolfe et al., 2002).
Cryopreservation protocols using these principles are available
for Q. robur; however, these methods generally result in high sur-
vival and normal root growth, but low recovery of the plumule
(Chmielarz et al., 2011). High sensitivity of plumule compared
with radicle tissues has been noted in several species (Pritchard
et al., 1995;Wesley-Sm ith et al.,2014) and is marked by deranged
intracellular organization in shoot tips upon thawing (Berjak et al.,
1999). Differences in radicle and plumule responsesto drying and
cryoexposure may arise from differences in drying rates (and so dif-
ferent watercontents during cryoexposure) or differences in sensi-
tivity to desiccation or desiccation– low temperature interactions.
Here, we used Quercus as a study system to investigate factors
presumed to influence the storagephysiology of recalcitrant seeds
and survival during cryoexposure. We tested the hypothesis that
species originating from environments with low rainfall produced
seeds with relatively greater tolerance to desiccation. We also
compared water relations and survival of radicle and plumule
tissues to test whether poor growth of shoots after cryoexposure
could be attributed to greater sensitivity to desiccation or to
greater susceptibilityto lethal ice formation.Desiccation tolerance
was quantified by the water content at which survival was nega-
tively impacted (i.e. damaging water content) or by the drying
time at which decreased viability was observed. This quantitative
treatment allowed us also to test the hypothesis that desiccation
tolerance correlates with survival following cryoexposure.
MATERIALS AND METHODS
Plant materials and viability assays
Our study considered four species of Quercus that have been clas-
sified in different subgenera. Species native to US temperate areas
are members of subgenus Quercus: Q. gambelii (white oaks,
section Quercus)andQ. rubra (black oaks, section Lobatae).
Species native to China are members of subgenera Quercus
[Q. franchetii (section Cerris)] or Cyclobalanopsis (Q. schott-
kyana, which is grouped with the Asian evergreen broad-leaf
oaks) (Table 1). The US oaks are from desert (Q. gambelii)and
mesic (Q. rubra) environments and the Chinese oaks are from
subtropical semi-humid areas. Two populations of Q. gambelii,
one from Wyoming (WY) and another from Nevada (NV),
were studied to provide a comparison between groups with and
without subfreezing winter temperatures (Table 1). Mature
fruits were collected and stored at 5 8C in plastic bags, punctured
to provide ventilation.
Most studies used excised embryonic axes. The excision pro-
cedure may induce a wounding response that complicates inter-
pretation of water-stress-induced responses. Freshly excised
axes were kept moist on blotter paper containing citric and ascor-
bic acid (0.2gL
–1
) solution until sufficient numbers were accu-
mulated for study (2 h). Viability of excised axes was assessed
by germination in vitro. Axes were surface-sterilized in 1 %
sodium hypochlorite for 10 min, rinsed twice in sterile water,
transferred to Woody Plant Medium with 0.3 % charcoal and
kept in darkness under room conditions for 48 h. After this
initial period, plates were transferred to 20 8C(Q. franchetii,
Q. gambelii and Q. rubra)or258C(Q. schottkyana) with a
light/dark photoperiod of 16/8 h. After 2 and 4 weeks we noted
whether axes had expanded, greened, formed callus or showed
normal development of roots and shoots, considered as doubling
TABLE 1. Collection and climate information for Quercus seed lots studied. Mean minimum and maximum temperature and total
rainfall are reported for October to February, months between shedding and germination for these species. Climate data for USA
locations (1961–90) and Chinese locations (1971 – 2000) were obtained from the World Wide Information Service (http://
www.worldweather.org/093/c00781.htm) and Climatic Data Centre (http://cdc.cma.gov.cn/), respectively
Species Collection location
Location
altitude (m)
Latitude and
longitude Collection date
Minimum
temperature (8C)
Maximum
temperature (8C)
Rainfall
(mm)
Q. franchetii Kunming, China 1900 25801′N, 102841′E 10 October 2012 5.64 17.14 164.4
Q. schottkyana Kunming, China 1900 25801′N, 102841′E 10 October 2012 5.64 17.14 164.4
Q. gambelii
(NV)
Kyle Canyon, Spring
Mountains, NV, USA
2075 36815′N, 115836′W 3 October 2012 4.818
.650
.3
Q. gambelii
(WY)
Crook County, WY,
USA
1646–1890 44859′N, 104856′W 15 September 2012 –7.06 6.377
.1
Q. rubra Delta and Schoolcraft
Counties, MI, USA
183–268 45844′N, 8783′W and
4286N
′,85838′W
8 October 2012 –8.52 0.52 348.5
Xia et al. — Stress tolerance of Quercus embryonic axesPage 2 of 13
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of radicle length or greening of shoots. A normal seedling exhib-
ited both root and shoot development. Drying and cryoexposure
treatments used aliquots of ten axes each and treatments were
repeated on two or three separate days; proportion data were
pooled to give sample sizes of 20– 30 axes according to
Crawley (2007).
Dehydration treatments and pressure– volume relationships
Embryos were dr ied rapidly over a stream of nitroge n gas (i.e.
‘flash drying’) for different durations (0– 1400 min) to adjust
water content. Fresh and dry masses were measured on three
to five individual embryos at each drying interval. In some
treatments, the axis was bisected at the cotyledonary node so
that the water contents of the plumule and radicle could be
determined separately. Sample dry mass was assessed after
heating tissues at 90 8C for 4 d. Water content (wc) was
expressed on a dry mass basis. Drying time course curves
were calculated from the relationship of time versus ln(
D
wc),
where
D
wc was wc at time t– final wc, which was determined
to be 0.05 g water g
21
dry mass under ambient room condi-
tions (30 % RH) in Fort Collins. The r
2
of these regressions
was usually .0.85.
The relationship between water content and water potential
was measured for embryonic axes, radicles, plumules and coty-
ledons using pressure– volume relationships. Plant materials
were soaked in polyethylene glycol (PEG, MW 8000) solutions
of concentrations between 0.1 and 1.15 g PEG g
–1
water at room
temperature for 2 d. Water contents were measured from fresh
and dry mass assessments as described in the previous paragraph.
Water potentials of PEG solutions were calculated according to
Michel (1983) and verified using a thermocouple psychrometer
(Decagon, Pullman, WA, USA). The accuracy of water content
measurements for axes soaked in the most concentrated PEG
solutions was verified with comparable water potential treat-
ments delivered using saturated salt solutions to control RH.
The pressure– volume curves were modelled from linear
regressions of water content and ln(water potential) calculated
separately for water potentials above and below – 6 MPa. All
relationships gave r
2
.0.94.
Tolerance of desiccation and cryoexposure
Desiccation tolerance of embryonic axes was assessed by elec-
trolyte leakage from reimbibing axes and survival and growth
following a drying challenge. Axes were flash-dried for different
durations and then slowly rehydrated on damp blotter paper for
1 h. These prehydrated embryos were soaked in 2 ml of distilled
water and electrical conductivity was measured every 5 min for
1 h using an ASAC 1000 (Applied Intelligence Systems,
Neogen, Lansing, MI, USA) conductivity meter. The slope of
the imbibition time versus conductivity relationship was used
to compare treatments and species. Measurements represented
the average of five replicates, each replicate consisting of two
axes.
After the 1 h soak, embryos were surface-sterilized and placed
on medium (described above) to assess survival and growth.
Embryos dried to non-lethal water contents were also chal-
lenged by exposure to liquid nitrogen using five cooling
methods: (1) bare axes injected individually into N
2
slush
(–2108C, formed by pulling a vacuum over liquid nitrogen)
using a spring-loaded desoldering pump (Elecon, Vallabh
Vidyanagar, India) with axes affixed to pins with a drop of gly-
cerol; (2) axes enclosed in 27 mg lightweight aluminium foil
packets (2 cm ×3 cm) (two axes per packet) and forcibly
plunged into N
2
slush with pre-cooled forceps; (3) axes enclosed
in 1.2 ml screw-cap polypropylene cryovials (ten axes per
cryovial) and submerged into liquid nitrogen; (4) the same cryo-
vial treatment placed in vapour above liquid nitrogen ( – 150 8C);
and (5) the same treatment as (4) except cryovials were placed in
a Styrofoam box packed with vermiculite for insulation and the
box was placed in vapour above liquid nitrogen. Temperature
within the core of the axis was measured in a subset of axes by
impaling them with fine-gauge (0.075 mm ×0.12 mm) bare
TABLE 2. Dry mass and water status of embryonic tissues of Quercus acorns prior to experimentation. Values are mean +s.d. (n.5).
Water potentials were calculated from the pressure– volume curves in Fig. 1. Pericarp dry mass was 40 % of cotyledon dry mass
among all species and pericarp water content was 0.11 and 0.41 g g
–1
for species from China and the USA, respectively. Arrows
under the cotyledon water potential represent the direction of water flow from cotyledon to axis () or from axis to cotyledon ()
Species
Dry mass (mg) Water content (g H
2
Og
–1
dry weight) Water potential (MPa)
Cotyledons Axis
Radicle and
plumule Cotyledons Axis
Radicle and
plumule Cotyledons Axis
Radicle and
plumule
Q. franchetii 580 +50 2.47 +0.51 0.99 +0.24 0.80 +0.15 0.95 +0.02 1.06 +0.01 –2.3–2
.9
a
–3.5
b
1.48 +0.51 0.88 +0.03 –3.4
b
–3.4
b
Q. schottkyana 530 +100 1.42 +0.31 0.22 +0.07 0.66 +0.04 1.06 +0.04 1.17 +0.03 –2.1–2
.8–2
.6
1.20 +0.31 1.03 +0.04 –2.7
Q. gambelii
(NV)
1130 +230 2.34 +1.32 nd 0.77 +0.05 1.20 +0.13 nd –2.7–3
.1nd
nd nd nd
Q. gambelii
(WY)
710 +370 2.38 +0.91 0.51 +0.23 0.85 +0.05 1.36 +0.14 1.34 +0.12 –3.2–2
.9–3
.0
1.87 +0.81 1.36 +0.15 –3.0
Q. rubra 1890 +540 4.13 +1.01 1.82 +0.52 0.69 +0.04 1.18 +0.12 1.23 +0.08 –3.2–2
.7–2
.6
2.52 +0.98 1.14 +0.18 –2.6
a
Measurement taken in November 2012.
b
Measurement taken in March 2013.
nd, not determined.
Xia et al. — Stress tolerance of Quercus embryonic axes Page 3 of 13
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wire, type T thermocouples (Omega Engineering, Stamford, CT,
USA). Temperature was recorded at millisecond intervals for
fully hydrated axes of all species. Additional experiments were
conducted using Q. rubra (the largest embryonic axis) to
measure the effect of water content on cooling rate. Axes were
flash-dried for 0, 45 and 120 min to obtain a range of water con-
tents. Cooling rates were calculated from the linear regression of
temperature and time for the temperature ranges between 5 and
–10, –40 and – 150 8C. Cooling rates for the narrower (i.e.
higher) temperature ranges were slower, probably as a result of
higher heat capacity. Only cooling rates between 5 and –40 8C
are reported in this study.
Axes were held at liquid nitrogen temperatures for 18 h prior to
viability assessments. Axes were warmed rapidly by immersing
them in a solution heated to 42 8C(Wesley-Smith et al., 2014);
we used a 0.5Msucrose solution to thaw axes.
Statistics
Statistical analyses were performed using R statistical soft-
ware (R Development Core Team, 2014). Water content data
were pooled within species for each drying time and differences
in drying rate among species or tissue types were assessed by
comparison of slopes calculated for water contents .0.15 g g
–1
.
Cooling rates for at least five axes per species and moisture
level were calculated from linear regressions of time versus tem-
perature between 5 and – 40 8C and fresh mass effects on cooling
rate were assessed by analysis of variance.
Survival and root and shoot development were treated as pro-
portion data and binomial distributions were used to calculate
error (Crawley, 2007). Desiccation tolerance was inferred by
damaging water contents that marked the points of deviation
from damaged to non-damaged physiology (Pammenter et al.,
1991;Farrant and Walters, 1998). We calculated the damaging
water content from the intersection of two lines: one line was
the lower limit of the confidence interval for unaffected viability
(a horizontal line drawn at mean survival – 1.5×the standard
deviation of the mean) and the other line was the regressed rela-
tionship between water content and survival at water contents
giving survival below the confidence interval for undamaged
physiology. The 90 % uncertainty range for damaging water
content was calculated from the intersection of lines drawn
from calculated slopes +the standard error of the slopes (i.e.
the 90 % confidence intervals of regression lines). We also
tested the feasibility of using the R library function for dose–
response to calculate the water content or drying time giving a
25 % reduction in viability or growth (LD75).
RESULTS
Size, water status and viability of axes prior to drying
and cooling challenges
Acorns from different sites and species varied in size (Table 2),
species from the USA tending to be larger or more variable than
species from China. Embryonic axes constituted between 0.12
and 0.33 % of total dry mass of the cotyledons. Upon receipt at
the National Center for Genetic Resources Preservation
(NCGRP), the water content of the cotyledons and embryonic
axes ranged from 0.66 to 0.85 g g
–1
and from 1.36 to 0.95 g g
–1
,
respectively, with Q. gambelii from Wyoming having the
highest water content and species collected from China tending
to be drier than US species.
0
0·5
1·0
1·5
2·0
2·5
0
0·5
1·0
1·5
2·0
2·5
C Q. gambelii (WY)
–12 –8 –4 0
D Q. rubra
0
0·5
1·0
1·5
2·0
2·5
0
0·5
1·0
1·5
2·0
2·5
A Q. franchetii
B Q. schottkyana
Water content (g H
2
O g
–1
d. wt)Water content (g H
2
O g
–1
d. wt)Water content (g H
2
O g
–1
d. wt)Water content (g H
2
O g
–1
d. wt)
Water potential (MPa)
Cotyledon
Plumule
Radicle
Axis
FIG. 1. Pressure– volume relationships of samples of Quercus embryonic axes
(solid circles), axis parts (open squares, plumules; open circles, radicles) and
cotyledon pieces (solid diamonds) osmotically dried using polyethylene glycol
(PEG, MW 8000) solutions of differing concentrations. Curves were calculated
from separate regressions of water content and ln(water potential) for water
potentials above and below –6 MPa, all relationships having r
2
.0.94.
Pressure– volume relationships quantified in these curves were used to convert
measured water content into water potential.
Xia et al. — Stress tolerance of Quercus embryonic axesPage 4 of 13
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Water potential (
C
w
) corresponding to the initial water content
of cotyledons and axes was interpolated from pressure– volume
relationships (Fig. 1), and ranged from – 2.1to –3
.2 MPa and
from –2.7to–3
.1 MPa, respectively (Table 2). Water content
in pressure –volume curves appeared typical of embryonic
tissues from other recalcitrant seeds (Farrant and Walters, 1998),
decreasing sharply with very small changes in water potential
TABLE 3. Initial viability and growth of embryonic axes, radicles and plumules of Quercus species and coefficients of drying rates modelled in Fig. 2. Percentage viability of
freshly excised axes was assessed by general appearance and growth of the root, shoot or both (i.e. a normal seedling), and the standard deviation, calculated from binomial
error distribution, is also indicated. Incidence of root growth compared with shoot growth was tested by a Tukey multiple comparisons of means test, with superscripted
letters indicating significance at the P,0.01 level. The drying rate model was calculated from the linear relationship between drying time tand ln(water content at t–0
.05
gH
2
Og
–1
dry weight). The intercept of the model was constrained by the average maximum water content for the tissue (given below); this value corresponds to the water
content of excised axes that have rested on damp blotter paper for 2– 3 h prior to drying treatments. The slope of the model is the rate coefficient and the standard error of
the coefficient is provided after the +symbol. Letter superscripts indicate significant differences among species for shoot growth or axis drying rate (P,0.05) and asterisks
represent significant differences between plumule and radicle as indicated
Species
Initial viability ((%) Maximum water content (g H
2
Og
–1
dry weight) Drying rate coefficient (min
–1
)
Expansion and
greening
Root or shoot
growth
Shoot
growth
Normal
seedling Axis Radicle Plumule Axis Radicle Plumule
Q. franchetii 86.3+5.581
.5+7.433
.3+3.3
b
33.3+3.32
.204 +0.560 2.112 +0.078 1.765 +0.155** 0.0196 +0.0013
a
0.0208 +0.0021 0.0265 +0.0022**
Q. schottkyana 95.5+5.994
.1+6.888
.8+9.1
a
79.4+21.31
.588 +0.476 1.528 +0.136 1.341 +0.227 0.0177 +0.0011
a
0.0179 +0.0014 0.0253 +0.0025***
Q. gambelii (NV) 97.4+4.495
.7+7.480
.9+11.7
a
80.9+11.71
.834 +0.472 nd nd 0.0078 +0.0006
b
nd nd
Q. gambelii (WY) 81.7+2.472
.5+3.52
.4+4.8
b
2.4+4.82
.443 +1.045 2.366 +0.998 2.654 +1.151 0.0078 +0.0007
b
0.0063 +0.0003 0.0101 +0.0012**
Q. rubra 98.7+2.698
.7+2.641
.7+19.8
b
41.7+19.81
.426 +0.585 3.006 +0.380 2.382 +0.419 0.0274 +0.0023
a
0.0297 +0.0042 0.0323 +0.0036
ns
Significance of differences between radicle and plumule drying rates: ns, not significant; **P,0.05; ***P,0.01.
nd, not determined.
Water content (g H2O g–1 d. wt)Water content (g H2O g–1 d. wt)Water content (g H2O g–1 d. wt)Water content (g H2O g–1 d. wt)
Drying time (min)
0
0·5
1·0
1·5
2·0
0 200 400
Q. rubra
0
0·5
1·0
1·5
2·0 Q. gambelii (WY)
0
0·5
1·0
1·5
2·0
Q. franchetii
0
0·5
1·0
1·5
2·0
Q. schottkyana
Whole axis
Plumule
Radicle
A
B
C
D
FIG. 2 . Dryingtime coursesfor samples of Quercus embryonicaxes (solidcircles)
and axis parts (open squares, plumules; open circles, radicles). Excised axes were
dried over a stream of nitrogen and divided above and below the cotyledonary
node to assess drying rate at different terminals. Curves were calculated from the
linear relationship between ln(water content – 0.05)anddryingtimewiththeinter-
cept constrained to ln(maximum water content) as given in Table 3(r
2
.0.9).
Xia et al. — Stress tolerance of Quercus embryonic axes Page 5 of 13
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at
C
w
.–6 MPa, and decreasing rather obliquely at
C
w
,
–6 MPa. For
C
w
.–6 MPa, water contents of embryonic axes
were higher than those of cotyledons. Despite having lower
water content, cotyledons of Q. franchetii,Q. schottkyana and
Q. gambelii (NV) had initial water potentials that were higher
than the respective axes and also higher than the initial water
potentials of Q. rubra and Q. gambelii (WY) cotyledons
(Table 2). No obvious differences in initial water potential were
noted among embryonic axes of different species and the water
relations of plumule and radicle portions were similar to each
other and to those of whole embryonic axes, except in
Q. franchetii (Table 2). The discrepancy for this species is attrib-
uted to a 4-month separation (November to March) between mea-
surements of whole axes and axis parts; during that storage period
embryo germination may have progressed. As water potentials
approached 0 MPa, plumulestended to have higher watercontents
than radicles in the species from the USA; however, the reverse
pattern appeared in the species from China (Fig. 1).
Freshly excised embryonic axes (i.e. no drying treatment)
showed high viability in culture, with at least 80 % of all axes
expanding and greening after 1 month (Table 3). Most axes
readily developed roots in culture; however, shoot development
did not occur as regularly (P,0.001 in paired t-test compari-
son), shoot growth being particularly rare in Q. gambelii
(WY). In other words, viability assessed by axis expansion and
greening was usually higher than viability assessed by normal
seedlings having both roots and shoots, even in control treat-
ments that received no water stress.
Drying treatment and response to water stress
Embryonic axes were dried over a stream of N
2
gas to nearly
ambient conditions (i.e. 30 % RH) within 3– 8 h depending on
species (Fig. 2). Axes from both Q. gambelii populations dried
more slowly than those of the other species (P,0.002) [the
drying time course for Q. gambelii (NV) is not given, but was
similar to data in Fig. 1CforQ. gambelii (WY)]. Plumule tissue
TABLE 4. Effects of drying on electrolyte leakage of Quercus
embryonic axes. Slope +standard error of slope, r
2
and
probability range of the correlation between rate of conductivity
increase in the leachate (data given in Fig. 4) and water content
achieved during drying are given. Water contents are interpolated
from the drying model coefficients provided in Table 3
Species
Conductivity in leachate (mA h
–1
mg
–1
H
2
O)
Slope r
2
Q. franchetii 23.7+4.90
.82**
Q. schottkyana 23.3+5.60
.71**
Q. gambelii (NV) 8.9+1.70
.72***
Q. gambelii (WY) 9.1+2.30
.53**
Q. rubra 9.3+2.30
.52**
**P,0.01; ***P,0.001.
Normal plantlet (%)
Drying time (min)
1400 1500
0
25
50
75
100
0 300 600
Viable (%)
0
25
50
75
100
Q. schottkyana
Q. franchetii
Q. gambelii (NV)
Q. gambelii (WY)
Q. rubra
B
A
FIG. 3 . Effect of drying time on viability (A) and normal plant growth (B) of
Quercus embryonic axes. Symbols indicate different species as indicated.
Curves were drawn as an aid to the eye.
0
10
20
30
40
0
10
20
30
40
0 0·5 1 1·5 2
Water content (g H2O g–1 d. wt)
Q. franchetii
Q. schottkyana
B
A
Q. gambelii (WY)
Q. gambelii (NV)
Q. rubra
Leakage rate (mA h–1 mg–1 d. wt)Leakage rate (mA h–1 mg–1 d. wt)
FIG. 4 . Electrolyte leakage from embryonic axes of Quercus that were flash-
dried to different water contents. Panel (A) shows the Chinese species,
Q. franchetii (solid circles) and Q. schottkyana (open circles); panel (B) shows
the US species, Q. gambelii (NV) (open triangles), Q. gambelii (WY) (open
squares) and Q. rubra (solid diamonds). Leakage rateswere positively correlated
with the watercontent to which embryonic axes were dried (P,0.01). The slopes
of the regressions are summarized in Table 4.
Xia et al. — Stress tolerance of Quercus embryonic axesPage 6 of 13
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tended to dry faster than the respective radicle tissue when drying
coefficients were compared among species (P,0.02) (Table 3).
Drying had variable effects on survival and organ develop-
ment depending on the species and duration of treatment
(Fig. 3). Slight drying had no effect on signs of viability (expan-
sion and greening of axes) (Fig. 3A) but appeared to stimulate
shoot growth in species exhibiting initially low plumule develop-
ment (Fig. 3B). As drying time increased, normal seedling devel-
opment declined and eventually signs of viability were also lost.
The drying time at which changes to the physiology of axes were
observed differed among species. Water contents corresponding
to drying times were calculated from drying time course models
(Table 3; Fig. 2) and are used henceforth to describe embryonic
axis responses to drying.
Leakage of electrolytes from rehydrating axes was used to
assess damage of embryonic axes following desiccation.
Conductivity of leachate increased as embryos were flash-dried
to progressively lower water contents (P,0.01 in regression
analyses for all samples) (Fig. 4; Table 4). Drying had a greater
effect on electrolyte leakage in the species from China, as indi-
cated by greater slopes of regression models: 23 mA h
–1
mg
–1
H
2
O for Q. franchetii and Q. schottkyana versus 9mAh
–1
mg
–1
H
2
O for Q. gambelii (NV), Q. gambelii (WY) and
Q. rubra.
50
75
100
0·0 1·0 2·0
Shoot development (%)
Water content (g H2O g–1 d. wt)
B
Root development (%)
Q. schottkyana
Q. franchetii
Q. gambelii (NV)
Q. gambelii (WY)
Q. rubra
A
0
25
50
75
100
0
25
FIG. 5 . Effect of drying to different water contents on normal root (A) and shoot
(B) growth of Quercusembryonic axes. Horizontal lines represent the proportion
of normal growth in axes before stress symptoms. Slanted lines on the left re-
present the regression of water content versus growth at lower water contents.
The intersection of the two lines was calculated as the damaging water content,
the point at which abnormal growth or organ mortality was observed with
further drying. Slanted lines on the right represent lower initial growth.
TABLE 5. Water contents limiting survival and growth of Quercus embryonic axes calculated using various approaches. Damaging water contents were calculated from
the intersection of horizontal and sloped lines as shown in Fig. 5; numbers in parentheses represent intersection of the horizontal line with the 90 % confidence interval for
the sloped lines. The water content and drying time giving a 25 % reduction in viability or growth (LD75) was calculated using the dose function from the R library;
standard error of the ‘dose’ giving 75 % of the maximum survival is also provided. The range of water contents corresponding to LD75 +standard error was calculated
from the drying time course function (Table 3) and is provided in parentheses beneath the LD75 drying time
Species
Damaging water content (g H
2
Og
–1
dw)
(90 % confidence interval) LD75 – water content (g H
2
Og
–1
dw) +standard error
LD75 – dry time (min) +standard error
(water content (g H
2
Og
–1
dw))
Expansion and
greening Root growth
Shoot
growth
Normal
seedling
Expansion and
greening Root growth Shoot growth
Normal
seedling
Expansion
and greening Root growth
Shoot
growth
Normal
seedling
Q. franchetii 0.40*
,†††
(0.30– 0.55)
0.56***
,†††
(0.53– 0.61)
0.37
ns,††
(0.35– 0.38)
0.57
ns,ns
(0.52–0.63)
0.72 +0.08
‡‡
0.92 +0.16
‡‡‡
1.86 +0.36
ns
1.98 +0.32
ns
69 +9
(0.66– 0.48)
43 +9
(1.03–0.73)
75 +14
(0.44– 0.24)
34 +18
(1.50– 0.76)
Q. schottkyana 0.36
(0.28– 0.48)
0.41
(0.38– 0.44)
0.45
(0.42– 0.49)
0.43
(0.40–0.47)
0.44 +0.04 0.64 +0.07 0.49 +0.05 0.8+0.07 83 +7
(0.42– 0.34)
54 +8
(0.68–0.51)
52 +7
(0.50– 0.38)
36 +8
(0.93– 0.70)
Q. gambelii
(NV)
0.19
(0.13– 0.32)
0.19
(0.14– 0.29)
0.38
(0.30– 0.52)
0.44
(0.32–0.70)
0.09 +0.24 0.39 +0.18 0.95 +0.12 1.04 +0.12 319 +36
(0.25– 0.16)
256 +32
(0.36–0.24)
205 +22
(0.48– 0.36)
121+21
(0.87– 0.64)
Q. gambelii
(WY)
0.13
(0.09– 0.22)
0.12
(0.09– 0.16)
0.23
(0.18– 0.34)
0.34
(0.22–0.72)
0.05 +0.12 0.38 +0.10 0.51 +0.09 0.70 +0.10 759 +84
(0.06– 0.05)
484 +65
(0.22–0.13)
360 +52
(0.16– 0.09)
287 +39
(0.41– 0.24)
Q. rubra 0.06
(0.05– 0.11)
0.08
(0.06– 0.11)
0.26
(0.22– 0.32)
0.23
(0.19–0.28)
0.19 +0.04 0.30 +0.05 2.24 +0.31 2.23 +0.32 263 +27
(0.05– 0.05)
263 +27
(0.06–0.05)
94 +17
(0.24– 0.11)
72 +17
(0.57– 0.25)
Correlations between damaging water content and LD75 calculated using water content among samples: ns, not significant; *P,0.1; ***P,0.01.
Correlations between damaging water content and LD75 calculated using water content among samples: ns, not significant;
††
P,0.05;
†††
P,0.01.
Correlations between damaging water content and LD75 calculated using water content among samples: ns, not significant;
‡‡
P,0.05;
‡‡‡
P,0.01.
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–100
–50
0
Temperature (∞C)
–200
–150
–100
–50
0
0246
0246
Time (s)Time (s)
Temperature (∞C)
Q. franchetii Q. rubra
Bare embryos
Q. franchetii
Q. schottkyana
Q. gambelii (WY)
Q. rubra
Q. rubra
0 min
120 min 45 min
0 min120 min 45 min
Insulated
container
Bare embryo
Foil packet
Bare embryos
into N
2
slush
N
2
slush
Cryovial
Cryovial
Vapour above LN Cryovials into
vapour above LN
0 200 400 600 0 100 3002000510
AC
BD
FIG. 6 . Cooling time courses of fully hydrated (A, B) and dried (C, D) embryonic axes of Quercus embryos. In (A), bare embryos of Q. franchetii,Q. schottkyana,
Q. gambelii (WY) and Q. rubra (see key) were plunged into N
2
slush and warmed in 40 8C water [only Q. gambelii (WY) is shown]. Panel (B) compares cooling time
courses of embryos of Q. franchetii cooled using various methods as indicated.Panels (C) and (D) give representativetime courses for Q. rubra embryos dried for 0, 45
and 120 min and then cooled in N
2
slush or in cryovials placedin vapour above liquid nitrogen-LN. The encircled discontinuities indicateice formation. Sample sizes
and statistics are summarized in Table 6.
TABLE 6. Cooling rates obtained by various methods of exposing Quercus embryonic axes to liquid nitrogen-LN. Cooling rates were
calculated from linear regressions of cooling time courses between +5 and – 40 8C. Values represent average +s.d. (n¼5). Cooling
rates of Q. gambelii (NV) were not measured because of the small size of the seed lot. Average fresh mass and water content of axes are
indicated. Cooling rates using different methods are significantly different at P,0.001 except the two vapour cooling treatments;
however, these treatments were significantly different when time to reach – 40 8C (i.e. the reciprocal of rate) was compared
Species
Water content
(g H
2
Og
–1
dry
weight)
Fresh mass
(mg)
Cooling rate between +5 and – 40 8C(8Cs
–1
)
Bare axis
plunged into
N
2
slush
(***
,†††
)
Foil packet
immersed in liquid
nitrogen-LN
(***
,†††
)
Cryovial
submerged in
liquid nitrogen-LN
(***
,†††
)
Cryovial placed in
vapour above liquid
nitrogen-LN (
ns,††
)
Insulated box placed
in vapour above
liquid nitrogen-LN
(
ns,nd
)
Q. franchetii 1.90 +0.11 5.66 +0.73 164 +18 55 +12 7.8+0.60
.36 +0.03 0.06 +0.01
Q. schottkyana 1.51+0.34 4.00 +0.37 170 +16 80 +98
.4+0.40
.40 +0.03 0.06 +0.01
Q. gambelii
(NV)
1.83 +0.47 7.35 +4.68 nd nd nd nd nd
Q. gambelii
(WY)
2.02 +0.63 5.87 +2.24 161 +28 56 +11 4.5+1.80
.56 +0.09 0.06 +0.01
Q. rubra 1.73+0.58 10.87 +2.27 98 +20 31 +11 2.8+0.70
.36 +0.08 0.05 +0.01
0.45 +0.13 6.17 +1.38 154 +18 61 +56
.2+0.80
.40 +0.02
0.15 +0.02 5.05 +1.43 181 +570+11 8.0+0.70
.51 +0.10
nd, measurement not done.
Correlations between cooling rate and fresh mass of fully hydrated axes: ns, not significant; ***P,0.001.
Correlations between cooling rate and fresh mass of Q. rubra at different water contents:
††
P,0.01;
†††
P,0.001.
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After no or mild stress, normal root development was observed
in 73 +4%[Q. gambelii (WY)] to 99 +3%(Q. rubra) of axes
(Fig. 5A). Approximately 93 +7, 82 +8 and 33 +3 % of axes
developed shoots in Q. schottkyana,Q. gambelii((NV) and
Q. franchetii, respectively, following no or mild water stress.
In contrast, mild stress appeared to increase shoot development
in Q. gambelii (WY) and Q. rubra, from initial low levels near
2 and 42 %, respectively (Table 3), to maximum levels of
64 +14 and 67 +12 %. Shoot and root development decreased
under severe water stress, and all signs of viability were lost in
axes that were dried for a prolonged period.
Desiccation tolerance among species and tissues was quanti-
fied by the water content at which loss of viability or reduced
1·1 0·8 0·6 0·4 0·2 0·1 1·1 0·8 0·6 0·4 0·2 0·1
0
20
40
60
80
100
1·8 1·2 0·8 0·6 0·4 1·8 1·2 0·8 0·6 0·4
Q. gambelii (NV)
0
20
40
60
80
100
1·20 0·90 0·40 0·20 0·10 0·08
Q. gambelii (WY)
1·20 0·90 0·40 0·20 0·10 0·08 1·20 0·90 0·40 0·20 0·10 0·08
0
20
40
60
80
100
1·1 0·8 0·6 0·4 0·2 0·1
Q. schottkyana
30 to 80 °C s–1
0
20
40
60
80
100
1·3 1·2 0·6 0·4 0·3 0·2 0·1
Q. rubra
1·3 1·2 0·6 0·4 0·3 0·2 0·1 1·3 1·2 0·6 0·4 0·3 0·2 0·1
3 to 8 °C s
–1 0·3 to 0·6 °C s–1
Water content (g H2O g–1 d. wt) Water content (g H2O g–1 d. wt) Water content (g H2O g–1 d. wt)
Survival (%)Survival (%) Survival (%)Survival (%)
Expanded/greened
Root
Shoot
Plant
No viability
nd
nd
nd
nd
nd
nd
nd
ABC
DE
FGH
IJK
FIG. 7 . Survival of embryonic axes of Quercus species at different water contents after exposure to liquid nitrogen. Samples were cooled using different methods to
achieve the range of cooling rates indicated above each column. Each row of graphs represents a different species or population. The bars show the percentages of the
recovery growth for survival (expansion and greening, dotted bars), normal development of roots (hatched bars), shoots (white bars) and regrowth of whole plants
(black bars). Stars represent treatments obtaining no survival, and ‘nd’ stands for treatments that were not studied. There was no survival in any treatment of
Q. franchetii (data not shown).
TABLE 7. Regression analyses using a general linear model
between damaging water content for survival (Table 3) and
maximum viability achieved after exposure to liquid nitrogen-LN
(Fig. 7) observed for Quercus species cooled at different rates
(Table 6)
Exposure method and average cooling rate Fd.f. Pr
2
Foil packet (30– 80 8Cs
–1
)55
.24 0
.005 0.948
Cryovial (3– 8 8Cs
–1
)24
.94 0
.016 0.892
Vapour phase (0.3–0.68Cs
–1
)10
.53
a
0.083 0.840
a
Vapour-phase treatments were not conducted for Q. gambelii (NV) because
there were too few seeds.
Xia et al. — Stress tolerance of Quercus embryonic axes Page 9 of 13
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growth was apparent (calculations are described in Materials and
methods). Damaging water content ranges were calculated from
the intersection of horizontal lines representing non-lethal stress
and sloped lines representing decreasing survival or growth with
decreasing water content (Pammenter et al., 1991;Farrant and
Walters, 1998) (Table 5). Representative graphs for root and
shoot growth are given in Fig. 5A, B and are similar in principle
to analyses for viability and normal seedling development, re-
spectively (data provided in Fig. 3A, B). Additional assessments
of desiccation tolerance used the dose–response function from
the R library to determine the water content or drying time corre-
sponding to a 25 % reduction in viability or growth (LD75)
(Table 5). The values for water contents varied among calcula-
tion approaches, but they were significantly correlated for expan-
sion and greening, root growth and shoot growth (for analyses of
damaging water content versus LD75 drying time but not for
LD75 calculated from water content measurements). The low
and variable incidence of shoot growth in unstressed axes
made LD75 calculations for shoot growth or normal seedlings
unreliable. In general, normal seedling growth (both root and
shoot development) was compromised during the early stages
of drying and preceded loss in the ability to develop either
organ. Ability to expand and green persisted to lower water con-
tents, but was eventually lost in all species. Plumules appeared to
have similar or greater tolerance than radicles in Q. franchetii and
Q. schottkyana; however, plumules were more sensitive towater
stress compared with radicles in Q. gambelii and Q. rubra. Based
on water content ranges that support viability and organ develop-
ment, the species can be ranked for desiccation tolerance as
Q. rubra .Q. gambelii (WY) .Q. gambelii (NV) .
Q. schottkyana .Q. franchetii.
Liquid nitrogen treatment and response to low-temperature stress
Interactions between water status and viability below 0 8Care
confounded by cooling rate, which affects the extent and cellular
location of freezing transitions. Cooling rate, in turn, can be
affected by specimen size, water content, containers, exposure
methods and temperature range (Walters et al., 2008). In our
hands, bare embryos plunged into N
2
slush cooled from room
temperature to – 200 8C in 1 – 2 s (Fig. 6A), while axes cooled
in an insulated box placed in vapour above liquid nitrogen
(Fig. 6B) reached – 150 8C after 1 –1.5 h. Cooling rates increased
as temperature decreased below – 80 8C, likely because the heat
capacity of the sample also decreased (data not shown).
Discontinuities in cooling time courses, indicative of freezing
events, were observable between – 10 and –30 8C when fully
hydrated samples (Table 3) placed in cryovials were cooled in
either liquid nitrogen or vapour above liquid nitrogen
(Fig. 6B). These discontinuities were not observed in more
rapidly cooled samples (Fig. 6B, C) or in samples dried and
cooled relatively slowly (Fig. 6D). Fresh mass, affected by
either axis dry mass or water content, had significant effects on
cooling rate in the faster cooling methods (i.e. plunging bare
embryos, foil packets or cryovials into liquid nitrogen; P,
0.01), but no effect was detected in embryos cooled relatively
slowly in the vapour above liquid nitrogen (Table 6).
Survival of axes following liquid nitrogen exposure was
assessed as a function of cooling method, axis water content
and species. For all species and water contents, plunging bare
axes into N
2
slush was mostly lethal; embryonic axes frequently
shattered and only a small percentage (,10 %) showed signs
of callus formation upon recovery (data not shown). A range of
responses was observed among water contents and species
when embryos were cooled in liquid nitrogen in foil packets
(cooling rate 30– 80 8Cs
–1
) or cryovials (3 – 8 8Cs
–1
) (Fig. 7).
Q. franchetii embryos at all water contents tested (0.13– 0.99 g g
–1
)
did not survive cryoexposure by any method (data not shown).
For other species, survival and organ development tended
to increase as the water content of cryoexposed axes decreased
to levels close to the damaging water content marking desicca-
tion damage (Table 5). Samples dried below damaging water
contents had low survival, presumably in response to
the desiccation treatment. Higher survival and more normal
development for a wider range of water contents was noted in
axes cooled at 3– 8 8Cs
–1
(cryovials plunged into liquid nitro-
gen) compared with 30–80 8Cs
–1
(foil packets containing
axes plunged into liquid nitrogen). Cooling at 0.3–0.68Cs
–1
by placing cryovials in the vapour above liquid nitrogen
reduced survival and organ development in axes of Q. rubra
(Fig. 7J, K), but gave survival results comparable to those
found with faster cooling treatments for Q. schottkyana and
Q. gambelii (WY) axes (Fig. 7B, C, G, H). We did not test
survival of axes exposed to liquid nitrogen using the slowest
treatment (0.06 8Cs
–1
).
A higher proportion of root compared with shoot development
was noted in recovering axes of all the species and cryoexposure
treatments (paired t-test, P,0.001). Overall, embryos of
Q. rubra and Q. gambelii were most amenable to cryopreserva-
tion treatments while axes of Q. schottkyana required a narrow
range of water contents and cooling rates for survival. The
highest four survival proportions for each species (Fig. 7) were
averaged (0 % for Q. franchetii) and regressed with damaging
water contents for expansion and greening given in Table 5
(Table 7). Significant relationships suggest that desiccation
tolerance and tolerance to cryoexposure are related.
DISCUSSION
Survival following desiccation and cryoexposure was measured
in embryonic axes of four Quercus species in order to compare
stress tolerance among species and among radicle and plumule
tissues within species. Our assessments of desiccation tolerance
by water contents that limit normal seedling development
(Table 5) are comparable to previous reports for Quercus
(Pritchard, 1991;Finch-Savage 1992;Sun, 1999;Ganatsas and
Tsakaldimi, 2013; P. Chmielarz, Institute of Dendrology,
Ko
´rnik, Poland and C. Walters, unpubl. res.) and other genera
(Pammenter et al., 1991;Berjak et al., 1993;Farrant and
Walters, 1998). The LD75 calculations presented here provide
some statistical advantages over the more typical damaging
water content analysis and are correlated with damaging water
contents if survival and growth are initially high. We show
here that pressure–volume relationships for embryonic axes of
diverse oaks are similar (Fig. 1), confirming that water content
measurements provide a reliable means to quantify and
compare water stress in excised axes of different species. Our
overall results are consistent with the general presumption that
seeds from all Quercus species are recalcitrant (Hong et al.,
1998;Dickie and Pritchard, 2002;Xia et al., 2012a) in that
Xia et al. — Stress tolerance of Quercus embryonic axesPage 10 of 13
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lethal effects of drying were observed and would preclude
freezer storage.
Increasingly, seed tolerance or sensitivity to desiccation is
regarded as a quantitative trait rather than a category describing
seeds that cannot be stored in the freezer. Ability to survive
and grow after low water stress is influenced by complex interac-
tions among phylogeny, growth conditions, maturity and post-
shedding conditions (Tweddle et al., 2003;Berjak and
Pammenter, 2008;Daws and Jensen, 2011). Here we also show
that these relationships are expressed differently in radicles and
plumules, depending on species (Fig. 5, Table 5). The higher
water contents of plumules compared with radicles at a given
water potential (compare pressure– volume curves in Fig. 1)
perhaps suggest that plumule cells were more vacuolated,
which can predispose cells to greater sensitivity to water loss.
To further understand the complex interactions leading to dif-
ferences in desiccation tolerance, we need reliable assays to
quantify the phenotype, which will involve precise control of
the duration and intensity of water stress and accurate measure-
ment of response. We believe that excising embryonic axes pro-
vides needed experimental control for comparative studies
among samples and tissues that vary in size (Table 2) and pres-
sure–volume relationships (Fig. 1). We show here that water
content assessments of the whole seed, containing diverse
tissues, may provide a relatively crude assessment of water
stress (Fig. 1) and response (Table 5) at the tissue level, and
that drying rate varies among species and tissue types (Fig. 1,
Table 3), such that drying time alone cannot be used to quantify
water stress. For these reasons, it is difficult to compare results
presented here with previous surveys of post-harvest physiology
of oaks in which the entire seed was used to assess bulk short-
term storage or recruitment of seedlings under natural conditions
(Olson, 1992;Bonner, 1996;Xia et al., 2012a;Joe
¨tet al., 2013).
We hypothesized that embryos from Quercus species adapted
to drier climates would exhibit greater tolerance to desiccation
compared with congeners growing in mesic environments.
Instead, we found that embryonic axes of the two Chinese sub-
tropical species from a semi-humid habitat were least tolerant
to desiccation, and a North American species adapted to cold,
wet winters was most tolerant to desiccation (Table 5).
Embryos of the desert-adapted North American species
Q. gambelii displayed intermediate desiccation tolerance; more-
over, the population originating from the temperate desert
(Wyoming) was more tolerant than the population originating
from the warm desert (Nevada).
Studies of the effect of growth environment on desiccation tol-
erance within species and among congeners consider the dur-
ation of both the maturation period (Vertucci et al., 1995;
Daws et al., 2004,2006) and the post-shedding, pre-germination
period (Dussert et al., 2000;Joe
¨tet al., 2013). Longer periods of
warmth during seed maturation allow seeds to accumulate more
dry matter (Daws et al., 2004,2006;Daws and Jensen, 2011),
which would also change pressure–volume relationships and
tend to decrease mechanical stress when water is removed from
cells (Walters and Koster, 2007). Therefore, one might hypothe-
size that seed dry mass positively correlates with desiccation tol-
erance (Daws et al., 2004,2006). However, there was no strong
correlation between damaging water contents (Table 5) and
axis or cotyledon dry mass (Table 2) in the samples studied
here (P.0.12). Seed survival after shedding may require
adaptive mechanisms to avoid desiccation stress over a long
dry period. Traits such as water permeability or water gradients
from cotyledon to axis may serve as protective mechanisms to
prevent water removal rather than provide tolerance of desicca-
tion (Farnsworth, 2000;Dussert et al., 2004;Hill et al., 2012;
Xia et al., 2012b;Joe
¨tet al., 2013). Anatomical adaptations of
the pericarp might help to resist water loss (Xia et al., 2012b)
and higher water potentials of the cotyledons compared with
axes [as seen in Q. franchetii,Q. schottkyana and Q. gambelii
(NV)] (Table 2) might provide a water reservoir for embryonic
axes. As can be expected, protection against water loss has
little advantage when water is actively removed, as in the
current study. Instead, the more desiccation-tolerant axes origi-
nated from populations that must cope with freezing tempera-
tures during winter (Table 1), and mechanisms that promote
water movement away from the embryonic axis would effective-
ly protect embryos from lethal freezing injury. For example,
Q. rubra seeds (Xia et al., 2012a) and axes (Fig. 2D) tended to
dry rapidly despite their large size, indicating a lack of effective
barriers to water loss. Moreover, initial water potentials of coty-
ledons from the more desiccation-tolerant axes [i.e. Q. gambelii
(WY) and Q. rubra] were lower than those of the axes (Table 2),
suggesting that the cotyledons did not serve as a water reservoir.
Here, we present the case that recalcitrant Quercus seeds may
exhibit desiccation avoidance or tolerance strategies depending
on whether they are adapted to non-freezing or freezing
winters, respectively. Intraspecific differences observed here
further suggest that survival mechanisms may be relatively
plastic and dependent on ecotype rather than species.
Survival following exposure to low temperature was tested
further for the purposes of developing cryopreservation strategies
for Quercus germplasm. In these studies, avoidance of lethal intra-
cellular ice formation was attempted bybalancing drying duration
and cooling rate (Wesley-Smith et al., 2004;Walters et al.,2008).
A preliminary assessment of water freezing transitions is provided
by cooling t ime courses, such as those gi ven in Fig. 6, showing dis-
continuities between – 10 and – 30 8Ci
nslower-cooled,fully
hydrated samples. More exact measurements of water freezing
events are gleaned from differential scanning calorimetry, which
did not detect transitions in axes having water contents less than
0.25 g H
2
Og
–1
dry weight (Vertucci, 1989).Samplesthat
tolerate drying below this so-called unfreezable water content
are also expected to tolerate cryoexposure. Consistently, axes of
Q. rubra and Q. gambelii (WY), which had damaging water con-
tents ≤0.25gg
–1
(Table 3), exhibited high survival ( .75 %) for
several moisture– cooling rate combinations (Fig. 6). Quercus
franchetii,Q. schottkyana and Q. gambelii (NV) had damaging
water contents near to or greater than typical unfreezable water
contents (Table 5) and these samples had low survival (,50 %)
following cryoexposure, likely because desiccation and freezing
damage could not be avoided simultaneously. The lowest water
content tested for axes of Q. gambelii (NV) was 0.4gg
–1
because of limited seed availability (Fig. 6); further drying may
boost survival in this sample and will be tested in subsequent
years. Cryoprotectants may be required to increase survival of
Q. franchetii and Q. schottkyana embryos, which had relatively
high damaging water contents.
Our results do not support the hypotheses that survival
increases with faster cooling in axes containing freezable
water. In fact, the highest survival was obtained in samples
Xia et al. — Stress tolerance of Quercus embryonic axes Page 11 of 13
at Lunds Universitet on October 21, 2014http://aob.oxfordjournals.org/Downloaded from
cooled at 3–8 8Cs
–1
(Fig. 7). The shattering of bare embryos
plunged directly into N
2
slush suggests to us that this rapid
cooling method imparted mechanical damage.
The results reported here (Fig. 7) are also consistent with a
growing number of reports in several species that embryonic
axes recovering from cryoexposure often fail to develop normally
(e.g. Berjaket al., 2011;Normah et al., 2011;Wesley-Smith et al.,
2014). In oak, roots may grow but development of shoots is
rare (Chmielarz, 1997;Gonza
´lez-Benito et al., 2002;Chmielarz
et al., 2011). Shoot growth from non-stressed axes was limited
after 4 – 6 weeks (Fig. 5), suggesting that plumule tissue may
have more fastidious growth requirements than radicles, and
hence shoots may have poorer survival after extreme stress.
Alternatively, the greater sensitivity to cryoexposure of plumule
relative to radicle tissues may be attributed to differences in desic-
cation tolerance, freezing tolerance and wounding effects result-
ing from excision. The combination of greater desiccation
sensitivity and faster drying of Q. gambelii (WY) and Q. rubra
plumules compared with radicles (Figs 2and 5; Tables 3 and 5)
should result in a relatively narrow optimum axis water content
and overall lower proportion of normal seedling development
(Fig. 7). Plumules and radicles of Q. franchetii and
Q. schottkyana were similarly sensitive to desiccation and more
sensitive than respective tissues from US species (Fig. 5,
Table 5), possibly explaining the consistently poor organ develop-
ment in these species following cryoexposure.
Conclusions
Comparisons of damaging water contents for survival and
growth in embryonic axes of four Quercus species suggested
that dry-adapted Quercus species do not produce the most
desiccation-tolerant seeds. Rather, strategies to survive freezing
temperatures during winter may contribute to innate desiccation
tolerance. In other words, desiccation tolerance is acquired
during embryo development and may be influenced by environ-
mental factors such as heat sum (sensu Daws), but additional
adaptations may involve freeze avoidance mechanisms during
winter. Embryonic tissues with greater tolerance to desiccation
had higher survival following cryoexposure. These inter-
relationships may explain why cryopreservation of recalcitrant
embryonic axes from temperate species is currently feasible,
and more research is needed to preserve germplasm from tropical
species (Walters et al., 2013). The results reported in this paper
also demonstrate faster drying and greater sensitivity of plumules
to desiccation compared with radicles, which may predispose
plumules to greater damage during cryoexposure. Overall,
these studies contribute to the understanding of the relationship
between geographical origin and seed post-harvest physiology
and demonstrate differential expression of desiccation sensitiv-
ity in mature embryo tissues.
ACKNOWLEDGEMENTS
We thank Professor Zhe-kun Zhou and Dr Jinjin Hu for providing
some of the seed material. We thank Adria DeCorte from the
Nevada Division of Forestry Southern Region, Las Vegas,
Nevada, for supplying the Nevada population of Q. gambelii.
Comments made on the original manuscript by Drs Marcin
Michalak (Institute of Dendrology, Ko
´rnik, Poland) and Daniel
Ballesteros (CREW, Cincinnatti Zoo and Botanical Garden,
OH, USA) helped to improve this article. This work was partially
supported by the National Natural Science Foundation of China
(grant 31200318 to K.X.), a Visiting Scholar Fellowship Grant
from the Chinese Academy of Sciences and the Independent
Research Program of the Chinese Academy of Sciences (grant
KSCX2-EW-J-24).
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