ORIGINAL PAPER
Cryopreservation of in vitro-grown shoot tips of Alnus glutinosa(L.) Gaertn.
M. Carmen San Jose • Silvia Valladares •
Laura V. Janeiro • Elena Corredoira
Received: 16 April 2013 / Revised: 30 August 2013 / Accepted: 9 September 2013 / Published online: 24 September 2013
� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2013
Abstract In vitro-grown shoot tips of Alnus glutinosa
(L.) Gaertn. were successfully cryopreserved by vitrifica-
tion. Shoot tips (0.5–1 mm) excised from 6-week-old
shoots were precultured in hormone-free Woody Plant
Medium (WPM) supplemented with 0.2 M sucrose, for
2 days at 4 �C in the dark, and then treated with a mixture
of 2 M glycerol plus 0.4 M sucrose, for 20 min at 25 �C.
Osmoprotected shoot tips were first dehydrated with 50 %
vitrification solution (PVS2), for 30 min at 0 �C, and then
placed in 100 % PVS2, for 30 min at 0 �C. The solution
was replaced with fresh 100 % PVS2, and the shoot tips
were plunged directly into liquid nitrogen. The shoot tips
were rewarmed in a water bath at 40 �C for 2 min, and then
washed twice, for 10 min at 25 �C, with 1.2 M sucrose
solution, before being transferred onto WPM supplemented
with 0.5 mg l-1 N6-benzyladenine, 0.5 mg l-1 indole-3-
acetic acid, 0.2 mg l-1 zeatin, 20 g l-1 glucose and 6 g l-1
Difco Bacto agar. The shoot tips were kept in darkness for
1 week and under dim lighting for another week, before
being exposed to standard culture conditions (16 h photo-
period). This protocol was successfully applied to three
alder genotypes, with recovery rates higher than 50 %.
Keywords Black alder � Long-term conservation �PVS2 � Shoot tips � Vitrification
Introduction
Alnus glutinosa is a medium-sized tree, of height 17–22 m
and diameter 60–70 cm. It has a shallow, highly branching
root system, especially when it grows in wet substrates. Its
roots develop nodules in symbiotic association with
Frankia alni, an endophytic nitrogen-fixing actinomycete.
The tree grows on riversides, in valley bottoms and wet
slopes, from sea level to an altitude of 1,700 m, and it
prefers acidic, silty substrates in which the water is fre-
quently renewed. The species is present throughout most of
Europe, Asia and the Northwest US. The wood, which is
almost white when freshly cut, burns slowly and is used as
a source of charcoal in the manufacture of gunpowder.
Since it stains well, it is also used in carpentry to imitate
noble woods such as ebony and mahogany, and in the
plywood manufacturing industry to make high quality
panels. The tannins present in the bark possess astringent
properties (antidiarrhoeic, local anticoagulant), and the
bark is used in home remedies as an antipyretic and local
analgesic (Menendez Valderrey 2006). Recent improve-
ments in markets for small diameter hardwoods and
development of markets for carbon sequestration have
increased the merchantability of woody nurse crops such as
alder (Bohanek and Groninger 2005).
At the beginning of the 1990s, a new disease caused the
loss of large numbers of alder trees. This disease was
associated with a previously unknown species of the fungus
Phytophthora (P. alni). This soil- and waterbone pathogen
causes aggressive root and collar rot on riparian alder
populations (Gibbs et al. 2003; Brasier et al. 2004). The
disease has been described in several European countries
with a destructive impact in Great Britain (Brasier et al.
2004; Jung and Blaschke 2004). All European alder species
and red alder (Alnus rubra) are highly susceptible,
Communicated by M. Capuana.
M. C. S. Jose (&) � S. Valladares � E. Corredoira
Instituto de Investigaciones Agrobiologicas de Galicia, CSIC,
Apartado 122, 15705 Santiago de Compostela, Spain
e-mail: [email protected]
L. V. Janeiro
INLUDES, Diputacion Provincial de Lugo, Ronda de la Muralla
140, 27004 Lugo, Spain
123
Acta Physiol Plant (2014) 36:109–116
DOI 10.1007/s11738-013-1391-x
A. glutinosa being the most susceptible to the pathogen.
The loss of the alder would lead to serious problems in
natural environments as the species plays a fundamental
role in stabilizing riverbanks, purifying water and con-
trolling the water temperature, and in the biodiversity of
terrestrial and aquatic habitats. Conservation of A. glutin-
osa resources is, therefore, worthy of research effort.
Germplasm preservation plays an important role in the
maintenance of biodiversity and avoidance of genetic
erosion. Advances in biotechnology have generated
opportunities for the conservation of genetic resources, and
the use and maintenance of plant materials at cryogenic
temperatures (cryopreservation) are now a suitable option
for long-term storage (Reed 2008; Kong and von Aderkas
2010). Cryopreservation allows the conservation of cells,
tissues and organs derived from in vitro culture (such as
shoot tips, callus cultures and somatic embryos) in liquid
nitrogen (Reed 2008). The main advantages of cryopres-
ervation are the simplicity and the applicability of the
technique to a wide range of genotypes (Engelmann 2004).
The method is based on the total arrest of cellular divisions
and metabolic processes as a result of storage at ultra-low
temperatures, usually that of liquid nitrogen (-196 �C)
(Niino and Sakai 1992). Theoretically, the plant material
can thus be stored unchanged for an indefinite period of
time (Engelmann 1997). Cryopreservation offers the most
efficient and cost-effective strategy for long-term storage
of genetic resources of vegetatively propagated plants and
is also increasingly recognized as an important tool for ex
situ conservation of the germplasm of endangered plant
species (Touchell 2000; Keller et al. 2008).
Our research efforts for the long-term conservation of
this species first focused on the development of a micro-
propagation protocol, using explants obtained from mature
adult trees (San Jose et al. 2013). Once this was accom-
plished, in this paper we studied the cryopreservation of
in vitro-grown shoot tips of mature alder using the vitrifi-
cation technique. To best of our knowledge, this is the first
report on the cryopreservation of alder species where shoot
tips are used. The only study that we have found on the
subject refers to the cryopreservation of seeds of A. glu-
tinosa (Chmielarz 2010). Shoot tips are often chosen as
explant because they contain a relatively small number of
undifferentiated cells, which are believed to maintain a
stable genetic content after recovery. Moreover, shoot tips
display more uniform genetic ploidy, which allows for
rapid regeneration into whole plantlets (Sakai 1995). The
rationale for cryopreserving shoot tips and meristems is
motivated by the need to conserve the germplasm of (1)
vegetatively proliferating species, (2) clonally maintained
elite genetic stocks, (3) species that produce recalcitrant
seeds, and (4) micropropagated plants of economic and
conservation significance (Benson and Harding 2012).
Vitrification refers to the physical process of transition of
an aqueous solution into an amorphous and glassy state
during ultra-rapid freezing. When the vitrification process
involves the cell cytosol, intracellular ice crystal formation
is hampered; thus, the tissue remains viable and responsive
to reintroduction to standard culture conditions (SCC). The
main advantages of the vitrification technique are: (1) the
explants are directly plunged into liquid nitrogen, which
simplifies the procedures considerably; (2) it is effective in
preserving the integrity of complex organs (such as shoot
tips), thus preventing partial destruction of meristems and
avoiding callusing. Hence, a normal regrowth of the pre-
served explants after rewarming is assured (Sakai 2000);
and (3) protocols of cryopreservation by vitrification are
highly repetitive (Lambardi and De Carlo 2003).
Materials and methods
Plant material and culture conditions
In vitro-grown shoots of alder (Alnus glutinosa (L.)
Gaertn.), derived from axillary buds excised from a 25–30-
year-old tree (Clone R4), were cultured on Woody Plant
Medium (WPM) (Lloyd and McCown 1980) supplemented
with 0.1 mg l-1 N6-benzyladenine (BA), 0.5 mg l-1
indole-3-acetic acid (IAA), 20 g l-1 glucose, and 7 g l-1
Difco Bacto agar. Medium pH was adjusted to 5.7 before
autoclaving at 121 �C for 20 min. The shoots were trans-
ferred to fresh medium every 3 weeks until completing a
subculture period of 9 weeks (San Jose et al. 2013). The
plant material was maintained in a growth chamber at
25 �C (day) and 20 �C (night) under a 16 h photoperiod,
with a light intensity of 50–60 lmol m-2 s-1, provided by
cool-white fluorescent lamps. These conditions were
defined as SCC.
Cryopreservation procedures
Effect of loading and vitrification solutions
In a preliminary experiment, the toxicity of the loading
solution was evaluated. Shoot tips (0.5–1 mm in
length 9 0.5–0.8 mm in width, and consisted of the apical
meristem and 2–6 leaf primordia) were excised from the
micropropagated shoots that had been cultured for 6 weeks
under the above described conditions and then precultured
on hormone-free WPM supplemented with 0.2 M sucrose
for 2 days at 4 �C in darkness. The shoot tips were trans-
ferred into 2 ml cryovials and treated with 1.6 ml loading
solution (LS) (2 M glycerol and 0.4 M sucrose; Matsumoto
et al. 1994) for 20 min at 25 �C. LS was removed and the
shoot tips were washed twice with unloading solution
110 Acta Physiol Plant (2014) 36:109–116
123
(1.2 M sucrose) for 10 min. The shoot tips were then
transferred to sterilized filter paper discs on the recovery
medium, consisting of WPM supplemented with
0.5 mg l-1 BA, 0.5 mg l-1 IAA, 0.2 mg l-1 zeatin (Z),
20 g l-1 glucose and 6 mg l-1 Difco Bacto agar, and
maintained in dark. After 1 day, the shoot tips were
transferred onto fresh medium without paper discs and
were incubated under the SCC. The shoot tips were
transferred to fresh medium every 2 weeks for 10 weeks.
In a second experiment, the toxicity of PVS2 solution
was tested. Precultured (0.2 M sucrose for 2 days) and os-
moprotected shoot tips were incubated in 1.6 ml PVS2
solution [30 % glycerol, 15 % ethylene glycol (EG), 15 %
dimethylsulphoxide (DMSO) and 0.4 M sucrose; Sakai
et al. 1990] at 0 �C for 60 min. The shoot tips were divided
into two groups. In one group, the PVS2 solution was
replaced with 0.6 ml of fresh solution and the cryovials
were then plunged directly in liquid nitrogen (LN) for at
least 2 h. The cryovials were removed from LN and rapidly
rewarmed in a water bath at 40 �C for 2 min. The other
group was not cryopreserved. In both groups, the PVS2 was
removed and cryopreserved and non-cryopreserved shoot
tips were washed twice with unloading solution for 10 min.
Recovery followed the procedures described above.
Preculture
The effect of the concentration of sucrose in the preculture
medium was examined. Shoot tips were precultured on
hormone-free WPM supplemented with 0.2 or 0.3 M
sucrose for 2 or 3 days at 4 �C in darkness. Control shoot
tips were not precultured. Loading, LN exposure, rinsing
and recovery followed the procedures described above for
both precultured and control shoot tips.
Efficiency of the two-step PVS2 exposure
Precultured (0.2 M sucrose for 2 days) and osmoprotected
shoot tips were dehydrated with a 50 % (half strength)
PVS2 solution, which consisted of 15 % (w/v) glycerol,
7.5 % (w/v) EG, 7.5 % (w/v) DMSO and 0.4 M sucrose,
and they were then dehydrated with 100 % (full strength)
PVS2 for 30, 45 or 90 min (15 min 50 % PVS2 ? 15 min
100 % PVS2, 30 min 50 % PVS2 ? 30 min 100 % PVS2
or 45 min 50 % PVS2 ? 45 min 100 % PVS2, respec-
tively) at 0 �C. In the control treatment, shoot tips were
dehydrated with 100 % PVS2 for 30, 60 or 90 min at 0 �C.
LN exposure, rinsing and recovery were as described
above.
The effect of the exposure temperature (0 or 25 �C) was
studied with 50 % PVS2 for 30 min ? 100 % PVS2 for
30 min. Preculture, loading, LN exposure, rinsing and
recovery followed the procedures described above.
Effect of the culture conditions during recovery
The effect of the light/dark regime during recovery of the
shoot tips was investigated. Preculture, loading, PVS2
exposure (50 % PVS2 for 30 min ? 100 % PVS2 for
30 min), LN exposure and rinsing steps were as before.
After rinsing, the shoot tips were either maintained for
1 day in darkness or maintained for 1 week in darkness
plus 1 week under dim lighting (25–30 lmol m-2 s-1)
before being transferred to SCC.
Effects of age of shoot tip donors
The effect of the age of shoot tip donors on survival and
recovery was tested. Shoot tips were taken from 3-, 6- and
9-week-old shoots cultured in vitro. The length of the
shoots was 0.8–1, 1–1.5 and 1.5–2 cm, respectively. Pre-
culture (0.2 M for 2 days), loading, 50 % PVS2 for
30 min ? 100 % PVS2 for 30 min at 0 �C, LN exposure,
rinsing and recovery (1 week dark ? 1 week dim light)
steps were as before.
Effect of the size of the shoot tips
Shoot tips (0.5, 1 and 2 mm in length, and consisted of the
apical meristem and 2–3, 4–6 and 7–8 leaf primordia,
respectively) were excised from 6-week-old shoots. Pre-
culture (0.2 M for 2 days), loading, 50 % PVS2 for
30 min ? 100 % PVS2 for 30 min at 0 �C, LN exposure,
rinsing and recovery (1 week dark ? 1 week dim light)
were carried out as described before.
Effect of genotype
The optimized protocol, defined from the experiments
described above, was tested in another two alder clones of
mature origin. Clone G1 was obtained from the base of a
tree aged 20–25 years, and clone R1 was taken from the
crown of a tree aged 25–30 years. Both clones were
established and maintained in vitro according to the con-
ditions indicated for clone R4.
Statistical analysis
Ten weeks after the shoot tips were plated on recovery
medium, the responses were assessed in terms of: (1) sur-
vival rates: the percentage of shoot tips exhibiting any sign
of growth, including callus formation, and (2) recovery
rates: the percentage of shoot tips that grew into new shoots.
In each experiment, three replicates Petri dishes with 10–12
shoot tips per dish (30–36 shoot tips in total) were used.
Each Petri dish was considered a single replicate in a
completely randomized block design. The influence of
Acta Physiol Plant (2014) 36:109–116 111
123
experimental factors was analyzed by a Student’s t test
(MedCalc version 10.3, Marekarke, Belgium). Differences
were considered significant at p B 0.05. The arcsine square
transformation was applied to percentage data prior to
analysis. Non-transformed data are presented in figures.
Results
Cryopreserved alder shoot tips turned blackish brown
within 1 day of thawing, but surviving shoot tips turned
green within 10–12 days on recovery medium, and suc-
cessfully recovered shoot tips resumed growth within
2–3 weeks. The first evident sign of shoot growth was leaf
development, followed by shoot development without
intermediate callus formation. After 10 weeks, the devel-
opment of the main shoot and 2–3 axillary shoots was
observed (Fig. 1). Surviving shoot tips that did not develop
into healthy shoots produced non-proliferating calluses.
Effect of loading and PVS2 solution
Ninety percent of the shoot tips survived when treated with
the loading solution, and the subsequent recovery rate was
81 %.
Treatment with the vitrification solution (PVS2) leads to
survival of 83 % of shoot tips, with a subsequent recovery
rate of 73 %. Immersion in LN reduced the survival and
recovery rates to 40 and 20 %, respectively. In the light of
these results, the following experiments were carried out
with the aim of improving these data.
Preculture
Shoot tips that did not undergo preculture (control) did not
survived immersion in LN. Excised shoot tips were
precultured with different concentrations of sucrose for 2
or 3 days to enhance osmotolerance to PVS2. Osmopro-
tection treatment with 0.2–0.3 M sucrose for 2 days was
significantly more effective for survival of explants after
LN immersion than treatment for a longer time (3 days)
(Fig. 2). In light of these results, a 2-day preculture treat-
ment period with 0.2 M sucrose in the medium at 4 �C in
darkness was used in all remaining experiments.
Two-step vitrification procedure
When the vitrification procedure was carried out in a single
step with 100 % PVS2, the best results were obtained after
exposure to the PVS2 for 60 min (Fig. 3). Thirty minutes
was not sufficient to dehydrate the shoot tips, and although
some shoot tips survived, there was no recovery. A two-
step dehydration procedure was examined as an alternative
to the use of 100 % PVS2 with the aim of enhancing the
recovery after cryopreservation. The two-step dehydration
procedure led to greater recovery than in the one-step
procedure, and the best rates were obtained when the shoot
tips were dehydrated with 50 % PVS2 for 30 min followed
by 30 min at 100 % PVS2. Dehydration in two steps at
0 �C for 60 min was, therefore, considered to be the best
treatment for cryopreservation of A. glutinosa shoot tips by
vitrification.
Shoot tips exposed to PVS2 at 25 �C (50 % PVS2 for
30 min plus 100 % PVS2 for 30 min) did not survive.
Fig. 1 Shoots developing from successfully cryopreserved shoot tips
of A. glutinosa (clone R4) 10 weeks after plating in recovery medium
0
10
20
30
40
50
60
70
80
90
Control 0,2 M 2 d 0,2 M 3d 0,3 M 2d 0.3 M 3d
Perc
enta
ges
Sucrose concentration (M) and preculture duration (days)
Survival
Recovery
a a
b
ab
a a
b
ab
b
b
c
b
ab
c
Fig. 2 Effect of preculture period and sucrose concentration on
survival and recovery of alder shoot tips (expressed as percentages).
Bars correspond to SE (standard errors) of means of three replicates.
In each treatment, values indicated with different letters are signif-
icantly different at p B 0.05
112 Acta Physiol Plant (2014) 36:109–116
123
Effect of the culture conditions during recovery
The survival and recovery rates were significantly enhanced
by maintaining the cryopreserved shoot tips for 1 week in
darkness and 1 week in dim light before transferring them
to SCC (Fig. 4) These conditions were, therefore, applied in
all remaining experiments.
Effects of age of donor plants on survival
The survival rate increased significantly as the age of the
shoot tip donors increased from 3 to 6 weeks (Fig. 5).
Recovery was also better in shoot tips from older donor
shoots (9 weeks) as compared to 3 weeks. At 6 weeks
there was a optimal growth and based on these results,
6-week-old shoots were used as the shoot tip donors in the
remaining experiments.
Effect of the size of the shoot tips
No significant differences were observed in shoot tips of length
between 0.5 and 1 mm (Fig. 6). However, the percentages of
survival and recovery were significantly lower in shoot tips of
length 2 mm. Shoot tips sized 0.5–1 mm were, therefore,
considered the most appropriate for alder cryopreservation.
Effect of genotype
Although the percent survival of the shoot tips differed
significantly between genotypes (clone R1 and R4), in all
cases it was greater than 50 %; the recovery rates did not
differ significantly (Fig. 7).
Discussion
We report here a protocol for the cryopreservation of alder
shoot tips by vitrification. Shoot tips are widely used in
0
10
20
30
40
50
60
70
80
90
30 min 60 min 90 min 15+15 min 30+30 min 45+45 min
Perc
enta
ges
100% PVS2 50% PVS2 + 100% PVS2
SurvivalRecovery
a aa
a
b
b
c
cc
de
a
Fig. 3 Survival and recovery of
vitrified alder shoot tips cooled
to -196 �C by two-step
vitrification procedure. Bars
correspond to SE (standard
errors) of three replicate
experiments. For each
treatment, values indicated with
different letters are significantly
different at p B 0.05
0
10
20
30
40
50
60
70
80
90
SCC 1w dark + 1w dim light
Per
cent
ages
Survival
Recovery
ab
a
b
Fig. 4 Effect of the culture conditions during the recovery of
cryopreserved alder shoot tips. Values are the means ± standard
errors of three replicate experiments. In each treatment, values
indicated with different letters are significantly different at p B 0.05
0
10
20
30
40
50
60
70
80
90
3 w 6w 9w
Per
cent
ages
Age of the donor of the shoot tips (weeks)
SurvivalRecovery
a
b
c
a
b b
Fig. 5 Effect of age of donor plants on survival and recovery of
cryopreserved A. glutinosa shoot tips. Bars correspond to SE of
means of three replicate experiments. In each treatment, values
indicated with different letters are significantly different at p B 0.05
Acta Physiol Plant (2014) 36:109–116 113
123
plant cryopreservation because of their genetic stability and
high rates of survival and regrowth (Zhao et al. 2005;
Senula et al. 2007).
Biological samples contain large amounts of water that
can cause mechanical damage to cells (due to the formation
of intra- and extracellular ice crystal during freezing and
thawing), and therefore, the water content of cells and
tissues must be reduced prior to cryopreservation (Fabian
et al. 2008; Yin and Hong 2009). Dehydration is a funda-
mental step in protecting cells from damage caused by the
extremely low temperatures of LN. Preculture of excised
shoot tips with different concentrations of sucrose prior to
loading treatment has been reported to be effective for
improving post-freezing survival rates of several species
(Vidal et al. 2005; Reed and Uchendu 2008; Sen-Rong and
Ming-Hua 2009; Chua and Normah 2011). Increased
sucrose concentration in the preconditioned medium leads
to accumulation of solute inside the cells, which maintains
the integrity of plasma and inner membranes during
dehydration and freezing (Plessis et al. 1993) and prevents
the formation of ice crystals during cooling and thawing
(Gropietsch et al. 1999). Ganino et al. (2012) demonstrated
that the concentration of solutes in the cells increased after
preculture of explants in media containing high concen-
trations of sucrose. In the present study, exposure of
explants for 2 days to 0.2 or 0.3 M sucrose enriched
medium at 4 �C yielded better recovery after vitrification
than exposure to the same medium for longer periods of
time (3 days).
Successful vitrification also requires careful control of
the highly concentrated vitrification solution to prevent
injury by chemical toxicity or excess osmotic stress during
dehydration (Fabian et al. 2008). Highly concentrated vit-
rification solutions yield the necessary degree of cyto-
plasmic dehydration to prevent formation of intracellular
ice, but they may also be cytotoxic if the exposure period is
prolonged (Sakai et al. 1990). Such cryoprotectant toxicity
causes cell injury in association with distinct ultra-struc-
tural changes, especially in the plasma membranes
(Fujikawa and Steponkus 1991; Steponkus et al. 1992).
Among the various vitrification solutions available, PVS2
is known to protect cryopreserved tissues effectively
without ice formation (Sakai et al. 1990). The optimum
exposure time to the vitrification solution and the concen-
tration of vitrification solution is weight-dependent and
species-specific (Niino et al. 1992). In alder, the recovery
rates were lower than 20 % in shoot tips exposed to 100 %
PVS2 solution for 60 to 90 min at 0 �C, and the results
were significantly improved (by more than 40 %) by
application of the two-step vitrification procedure for
60–90 min at 0 �C. The two-step vitrification procedure
has been evaluated by various authors. Takagi et al. (1997)
reported that exposure of shoot tips of taro (Colocasia
esculenta) to 60 % PVS2 for 20 min at 25 �C, followed by
100 % PVS2 for 10 min at 25 �C yielded the highest rate
of survival (77 %). Jiwu et al. (2007) reported that the use
of a cryoprotective solution of 60 % PVS2 for 50 min at
room temperature followed by 100 % PVS2 for 30 min at
0 �C yielded a 52.6 % regeneration rate in papaya. Kaity
et al. (2008) reported a 61–73 % regeneration rate when
papaya shoot tips were pretreated with 20 % PVS2 for 1 h
at room temperature and then 100 % PVS2 for 20 min at
room temperature. Sen-Rong and Ming-Hua (2009)
reported survival rates of 79–84 % for shoot tips of
Emmenopterys henryi, which were dehydrated with 60 %
PVS2 solution for 30 min at 0 �C and then 100 % PVS2
for 40 min at 0 �C. Careful control of the dehydration
0
10
20
30
40
50
60
70
80
90
0.5 mm 1 mm 2 mm
Per
cent
ages
Shoot tips size
Survival
Recovery
a
a
b
a
a
b
Fig. 6 Effect of shoot tip size (0.5–2 mm) on survival and recovery
of alder cryopreserved shoot tips. Bars correspond to SE of means of
three replicate experiments. In each treatment, values indicated with
different letters are significantly different at p B 0.05
0
10
20
30
40
50
60
70
80
90
Clone G1 Clone R1 Clone R4
perc
enta
ges
SurvivalRecovery
ab a
b
aa
a
Fig. 7 Effect of genotype on survival and recovery of alder
cryopreserved shoot tips. Bars correspond to SE of means of three
replicate experiments. In each treatment, values indicated with
different letters are significantly different at p B 0.05
114 Acta Physiol Plant (2014) 36:109–116
123
procedure appears to be essential for the successful cryo-
preservation of plant tissues by vitrification (Panis et al.
2001).
Post-cryopreservation conditions are also critical factors
in the establishment of a cryopreservation protocol. In this
study, shoot tip recovery was positively influenced by
incubation of cryopreserved explants in the dark after
rewarming, followed by a gradual transfer to light. Similar
conditions were used in the cryopreservation of Tamarix
boveana (Cano-Castillo and Casas 2012). Various authors
have indicated that the best rates of survival after cryo-
preservation were achieved by incubating cryopreserved
material in dim light or total darkness (Touchell et al. 2002;
Gonzalez-Arnao and Engelmann 2006; Gonzalez-Arnao
et al. 2009). Increased survival under these conditions has
been attributed to the damage repair that may take place in
darkness (Sen-Rong and Ming-Hua 2009).
The size and developmental stage of the cryostored
material must be optimal to ensure high post-thaw shoot
recovery rates (Takagi 2000). The developmental stage of
the plant material directly determines the effect of cryo-
preservation (Li et al. 2009). Suitable physiological status
and appropriate growth conditions of shoot tip donor plants
may be key factors in the tolerance to LN treatment during
the cryopreservation protocols (Takagi et al. 1997). In the
present study, the highest recovery rates were obtained in
shoot tips derived from 6-week-old donor shoots. This
suggests that physiological conditions are very important
for increasing the tolerance to cryogenic procedures.
Regarding the size of the shoot tips, the best results in alder
were obtained for shoot tips of length 0.5–1 mm which
consists mostly of meristematic cells and the use of larger
shoot tips had a negative effect on survival and recovery.
Vidal et al. (2005) reported that smaller apices tend to
consist of a homogeneous population of small, actively
dividing cells with few vacuoles. These characteristics
make the shoot tips more tolerant to dehydration than
highly vacuolated and differentiated cells, which form part
of large apices. In contrast, longer shoot tips (2–3-mm
long) have been reported to survive and regrow after vit-
rification and cryostorage in other species such as Morus
alba (Arias Padro et al. 2012), Vitis vinifera (Shatnawi
et al. 2011), Passiflora suberosa (Garcıa et al. 2011), and
Pinus kesiya (Kalita et al. 2012), if dehydration treatment
was appropriated.
The success of plant germplasm cryopreservation is
genotype-dependent (Reed et al. 2000). From a practical
point of view, it is important to establish protocols that are
appropriate for several genotypes (Ryynanen and Haggman
2001). The protocol defined in the present study yielded
moderate shoot recovery rates in all genotypes tested, and
because of the homogeneity obtained from the post-LN
survival rates of three different genotypes of A. glutinosa,
we conclude that the proposed protocol is appropriate for
cryopreservation of this species, which is currently under
serious threat from attack by P. alni.
Author contribution MC San Jose and E Corredoira
designed the research, analyzed the data and wrote the
paper. S Valladares and LV Janeiro conducted the research.
All authors have read and approved the final manuscript.
Acknowledgments The authors thank Dr. A.M. Vieitez for useful
advice and suggestions and Carlos Suarez for technical assistance.
This study was funded by INLUDES (Diputacion Provincial de
Lugo).
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