2. 100
highly valuable for ornamental purposes, reforestation, timber and plywood production.
However, its conservation is problematic due to its low rooting capacity, shoot dormancy, low
seed germinability (about 10%, 2) and low seedling viability (4, 7, 15).
In vitro techniques are widely used for multiplication and conservation of species whose
propagation and storage by classical techniques is problematic (8, 21), such as S.
sempervirens (21). For medium-term conservation, in vitro slow-growth storage is employed,
whereas cryopreservation protocols are used for long-term conservation (13). Recently, an in
vitro slow-growth protocol was established for this species, which allowed conserving S.
sempervirens shoot cultures at 4°C in the dark for up to 15 months, with 60% regrowth (21).
However, to our knowledge, no cryopreservation protocol is available for S. sempervirens.
Today, vitrification-based cryopreservation protocols are available for a large number of
plant species, originating both from tropical and temperate climates. Among the seven
vitrification-based procedures available, encapsulation-dehydration, vitrification,
encapsulation-vitrification and droplet-vitrification are the most commonly employed (10,
26). The encapsulation-dehydration procedure is based on the technology developed for the
production of artificial seeds (6). Explants are encapsulated in alginate beads, pregrown in
liquid medium enriched with sucrose for 1 to 7 days, partially desiccated in the air current of a
laminar air flow cabinet or with silica gel to a water content around 20% (fresh weight basis),
then cooled rapidly. This technique is applied to apices as well as to cell suspensions and
somatic embryos of numerous species (11). Vitrification involves treatment of samples with a
loading solution containing cryoprotectants at intermediate concentration, usually 2 M
glycerol + 0.4 M sucrose (17), dehydration with highly concentrated vitrification solutions,
usually the PVS2 (30) and PVS3 solutions, (20), rapid cooling and rewarming, removal of
cryoprotectants in an unloading solution and regrowth. This procedure was developed for
apices, cell suspensions and somatic embryos of numerous species (29, 31). Encapsulation-
vitrification is a combination of encapsulation-dehydration and vitrification procedures, where
samples are encapsulated in alginate beads, then subjected to vitrification solutions. It has
been applied to apices of an increasing number of species (31). The droplet-vitrification
technique was applied to a number of species including potato, asparagus and apple apices
(31). Apices are pretreated with vitrification solution, then placed on an aluminium foil in
minute droplets of vitrification solution and cooled rapidly in liquid nitrogen (LN). The main
advantage of this technique is the very high cooling and warming rates achieved, thanks to the
small volume of cryoprotectant solution employed and to the fact that explants are plunged
directly in liquid nitrogen for cooling and in unloading medium for rewarming. This avoids
the buffering effect of the cryotube and of the relatively large volume of cryoprotectant
solutions employed in other vitrification protocols.
This study aimed at determining a cryopreservation protocol for apical and basal buds
sampled from S. sempervirens in vitro shoot cultures. Three techniques were compared:
vitrification, encapsulation-vitrification and droplet-vitrification. Histo-cytological
observations were performed to observe the effect of the successive steps of the
cryopreservation protocol on the structural integrity of the buds.
MATERIALS AND METHODS
Plant material
In vitro shoot cultures of S. sempervirens (D. Don.) Endl. were initiated from an old
elite tree, growing in a public garden of Florence (Italy).
3. 101
Standard culture conditions
Cultures were maintained by periodic transfers (4-week intervals) of 1-1.5 cm apical
shoots on semi-solid Murashige and Skoog medium (MS, 18), with 1 mg l-1
N6
-benzyladenine
(BA), 30 g l-1
sucrose and gelled with 7 g l-1
agar (MS1, regrowth medium). The pH was
adjusted to 5.8 with 1N NaOH or HCl before addition of agar and the medium was autoclaved
for 20 min at 121°C. Cultures were incubated at 23±2°C under a 16 h light/8 h dark
photoperiod, with a light intensity of 36.3 µmol m-2
s-1
provided by cool daylight fluorescent
lamps.
Preliminary experiments
Prior to cryopreservation trials, experiments were performed to study the effect of
cold-hardening of in vitro shoot cultures and of sucrose preculture on regrowth of apical and
basal buds.
Cold-hardening of in vitro shoot cultures
Shoot cultures were transferred to 4°C in the dark 14 days after the last subculture and
cold-hardened for 0, 1, 2, 3 or 4 weeks. Following cold-hardening, apical and basal buds (~
0.5 cm) were excised aseptically and transferred to one of the following media (all with 30 g l-
1
sucrose and gelled with 7 g l-1
agar) under standard culture conditions: (i) MS0: hormone-
free MS medium; (ii) MS1: MS medium with 1 mg l-1
BA; (iii) MS + AC: hormone-free MS
medium with 20 g l-1
activated charcoal; (iv) QL0: hormone-free Quoirin and Lepoivre
medium (QL, 24); (iv) QL1: QL medium with 1 mg l-1
BA; and (vi) QL + AC: hormone-free
QL medium with 20 g l-1
activated charcoal. Survival and regrowth of buds were recorded
after 4 weeks.
Sucrose preculture of apical and basal buds
Following cold-hardening of in vitro shoot cultures at 4°C for 4 weeks, apical and
basal buds were excised and transferred to QL medium with 0.12, 0.25 or 0.50 M sucrose, and
cultured for 24, 48 or 72 h under standard conditions. They were then transferred to QL + AC
medium and subcultured at 4 week intervals. Regrowth of buds was recorded at the end of the
first subculture period.
Cryopreservation of apical and basal buds using vitrification-based techniques
Three different vitrification-based techniques were compared for cryopreserving S.
sempervirens apical and basal buds: (i) vitrification, (ii) encapsulation-vitrification and (iii)
droplet-vitrification. For all cryopreservation experiments, apical and basal buds were
sampled from shoot cultures that were cold-acclimated for 4 weeks at 4°C in the dark on QL
+ AC medium. Buds were precultured for 48 h on QL medium with 0.12 M sucrose (apical
buds) or for 72 h on QL medium with 0.25 M sucrose (basal buds), then treated as described
below.
PVS2 vitrification
Apical and basal buds were transferred to 2 ml Nalgene®
cryovials (14-15 buds per
cryovial) and incubated in loading solution (LS; 2 M glycerol + 0.4 M sucrose in liquid MS
medium; 17) for 30 min at 25°C. LS was then replaced with PVS2 vitrification solution (30%
glycerol (w/v), 15% ethylene glycol (w/v), 15% DMSO (w/v) in MS medium with 0.4 M
sucrose, 30) and buds were treated with PVS2 for up to 180 min at 0°C. Following the PVS2
treatment, half of the apical and basal buds were suspended in 0.6 ml fresh PVS2 and directly
immersed in LN, while the other half (controls) were washed in unloading solution (liquid MS
medium with 1.2 M sucrose, 30) for 20 min at 25°C, then plated on QL + AC medium under
4. 102
standard conditions for regrowth. At the end of the first 4-week subculture, buds were
transferred to MS1 medium for induction of multiple buds/shoots. After at least 1 h storage in
LN, cryopreserved samples were rewarmed in a water-bath at 40°C for 2 min, then treated as
control samples.
Encapsulation-vitrification
Apical and basal buds were suspended in a 3% sodium alginate (low viscosity, 200
cps) solution and dropped in 100 mM CaCl2 solution, each drop containing one explant (14).
The beads were kept for 25 min at room temperature in the CaCl2 solution to ensure complete
polymerization of calcium alginate, collected on a sterile sieve and washed with sterile
distilled water. Beads were transferred to 2 ml Nalgene®
cryovials (5-6 beads per cryovial)
and incubated in LS for 30 min at 25°C. LS was replaced with PVS2 and buds were treated
with PVS2 for up to 180 min at 0°C. Half of the beads were washed in unloading solution
(controls) for 20 min at 25°C and the other half cryopreserved by direct immersion in LN.
Samples were then treated as described above for the vitrification technique.
Droplet-vitrification
Sterile aluminium foil strips (~ 5 x 15 mm) were placed in an open Petri dish, resting
on a frozen cooling element (temperature around 0°C), and three 4-5 µl drops of PVS2 were
dropped on each aluminium foil strip. Apical or basal buds were placed in the PVS2 drops
(one bud per drop), and treated for up to 180 min at 0°C. After PVS2 treatment, half of the
buds (controls) were immersed in unloading solution for 20 min and plated on QL + AC
medium for regrowth. The other half of the explants, the aluminium foils were plunged in 2
ml Nalgene®
cryovials previously filled with LN, which were then transferred to LN tanks for
storage. Rewarming was performed at room temperature by retrieving the aluminium foils
from LN and immersing them in the unloading solution for 20 min at 25°C. Buds were then
transferred on QL + AC medium and incubated under standard culture conditions for
regrowth.
Histological observations
Apical and basal buds, sampled at several stages of the cryopreservation protocol were
fixed in 2% glutaraldehyde and 2% formaldehyde (buffered with 0.05 phosphate buffer at pH
7.2) at 4°C for 24 h. They were dehydrated for 24 h at 4°C in ethylene glycol monomethyl
ether, then in absolute ethanol and embedded in LKB historesin according to the procedure of
Yeung and Law (33). Two m-thick sections, obtained after sectioning with a glass knife,
were stained using the Periodic Acid-Schiff reaction and counter-stained with Amido Black
10B (27).
Data collection and statistical analysis
Two replicates of 30 apical and basal buds were used for each treatment of
cryopreservation experiments and all experiments were repeated at least three times. Only
buds not showing any symptom of contamination were considered for data evaluation.
Regrowth (% of buds which had elongated and/or from which new buds developed) was
evaluated 4 weeks after placing the buds on regrowth medium.
Statistical analysis of percentages was carried out by a non-parametric, X2
-based test,
the post hoc Multiple Comparisons test (16). Data were subjected to ANOVA, followed by
the least significant difference (LSD) test at P ≤ 0.05 to compare means.
5. 103
RESULTS
Effect of shoot culture cold-hardening on regrowth of apical and basal buds
Table 1 presents the results of the ANOVA performed on the effect of bud type,
medium composition and cold-hardening on S. sempervirens regrowth. All interactions
studied had a significant effect, except that between bud type, culture medium and cold-
hardening.
Table 1. Multifactorial ANOVA of experiment on the effect of bud type, medium composition
and cold-hardening on regrowth of S. sempervirens buds. Before performing ANOVA,
percentage data were subjected to arcsin % transformation (NS, not significant, **
significant at P ≤ 0.01, *, significant at P ≤ 0.05).
Source df Mean-square F-ratio P
Bud type 1 10879.6 63.2 **
Medium 5 19660.5 114.1 **
Cold-hardening 4 4785.4 27.8 **
Bud type x medium 5 381.6 2.2 *
Bud type x cold-hardening 4 3173.1 18.4 **
Medium x cold-hardening 20 1454.0 8.4 **
Bud type x medium x cold-hardening 20 267.0 2.5 NS
Error 240 172.3
Table 2. Effect of bud type, culture medium and cold-hardening (storage at 4°C in the dark)
on regrowth (%) of S. sempervirens buds. For each treatment, different letters indicate
significant differences at P≤0.01. (MS0= MS, hormone-free; MS1= MS+ BA; QL0= QL,
hormone-free; QL1= QL + BA; AC= Activated Charcoal).
Treatment Regrowth (%)*
Bud type
Apical buds
Basal buds
33.9 A
16.0 A
Culture medium
MS0
MS1
MS + AC
QL0
QL1
QL + AC
36.2 B
5.2 D
47.6 B
18.0 C
0.2 E
68.8 A
Cold-hardening
0 (control)
1 week
2 weeks
3 weeks
4 weeks
50.1 A
21.4 B
14.1 B
21.3 B
19.0 B
* Percentage data were subjected to arcsin % transformation before analysis by ANOVA,
followed by LSD test at P ≤ 0.01.
6. 104
The bud type (apical or basal) employed had no effect on regrowth (Table 2). Addition
of BA in the culture medium was detrimental to regrowth with both mineral solutions. When
no growth regulators were present in the medium, regrowth was intermediate. Addition of
activated charcoal in QL medium led to highest regrowth. Cold-hardening always reduced
regrowth, whatever the duration of cold-hardening treatment employed.
Effect of sucrose preculture on regrowth of apical and basal buds
All interactions studied between bud type, sucrose concentration and preculture
duration had a significant effect on S. sempervirens regrowth, except that between bud type,
preculture duration and sucrose concentration (Table 3).
Table 3. Multifactorial ANOVA of experiment on bud type, sucrose concentration and
preculture duration on regrowth of S. sempervirens buds. Before performing ANOVA,
percentage data were transformed by arcsin % (NS, not significant, ** significant at P ≤
0.01, *, significant at P ≤ 0.05).
Source df Mean-square F-ratio P
Bud type 1 2423.3 14.6 **
Duration 2 1412.6 8.5 **
Sucrose 2 6654.4 40.0 **
Bud type x duration 2 1637.4 9.9 **
Bud type x sucrose 2 650.5 3.9 *
Duration x sucrose 4 801.3 4.8 **
Bud type x duration x sucrose 4 371.6 2.2 NS
Error 90 166.2
Table 4. Effect of bud type, sucrose preculture duration and sucrose concentration on
regrowth (%) of S. sempervirens buds. For each treatment, different letters indicate
significant differences at P≤0.01.
Treatment Regrowth (%)*
Bud type
Apical buds
Basal buds
76.5 A
61.4 B
Sucrose preculture duration
24h
48h
72h
63.9 A
62.6 A
63.5 A
Sucrose concentration
0.12M
0.25M
0.50M
83.1 A
78.7 A
26.1 B
* Percentage data were subjected to arcsin % transformation before analysis by ANOVA,
followed by LSD test at P ≤ 0.01.
7. 105
Table 4 shows the effect of bud type, sucrose preculture duration and sucrose
concentration on regrowth of S. sempervirens buds. Apical buds had a significantly higher
regrowth than basal ones. Sucrose preculture duration had no significant effect on regrowth,
while lower sucrose concentrations induced higher regrowth.
Cryopreservation of apical and basal buds
Among the three techniques tested, vitrification, encapsulation-vitrification and droplet-
vitrification, only the latter resulted in survival and regrowth after cryopreservation under the
experimental conditions tested.
With apical buds, regrowth of controls dropped from 78% after 15 min PVS2 exposure to 5%
after 180 min exposure (Fig. 2A). After cryopreservation, regrowth was possible for PVS2
exposure durations comprised between 90 and 180 min but it remained low, with a maximum
of 18% after 135 min treatment.
Figure 1. Response of apical (A) and basal buds (B) to PVS2 treatment (control) or to PVS2
treatment and storage in LN (LN +) for droplet-vitrification. In A and B, droplet-vitrification
was applied after a 4 week cold-hardening and 48 h preculture with 0.12 M sucrose (apical
buds) or 72 h preculture with 0.25 M sucrose (basal buds).
A A
B B
B B
B
BCBC
C
a
aa a
b bb C C
ab
a
a
aa
bbb
a
bb
A
A
A
A A A
AB
B B
B B
B
A
B
8. 106
Figure 2. Cryopreservation of Sequoia sempervirens. Shoot tips, placed inside PVS2 drops
on aluminium foils (A, bar, 2 cm). Regrowth of an apical (B, bar, 1 cm) and basal (C, bar, 1
cm) bud after PVS2 treatment and exposure to LN.
With basal buds, regrowth of control explants also decreased regularly, but more
slowly compared with apical buds, reaching 18% after 180 min PVS2 exposure (Fig. 2B).
After cryopreservation, regrowth was possible over a larger range of PVS2 treatment
durations, between 30 and 180 min. The highest regeneration percentage was slightly higher
(22%) than that measured with apical buds, and was also achieved after 135 min PVS2
exposure.
Histological observations
S. sempervirens apical and basal buds employed for cryopreservation consisted of a
meristematic dome covered by several leaf primordia. After PVS2 treatment for 105 min,
almost all cells of the meristematic dome and of the first leaf primordia of surviving buds
were able to maintain their integrity (Fig. 3A). These viable cells (especially in the four-five
external layers of the meristematic dome) had a dense cytoplasm, small vacuoles, an intact
nucleolus, and contained large quantities of sugar. Cells of the more external leaf primordium,
had a larger size and contained more numerous and larger vacuoles. Cells, which did not
withstand the PVS2 treatment, appeared sallow. Similarly, almost all the cells of buds, which
did not withstand PVS2 treatment, were relatively large (Fig. 3B). These cells tended to swell
and collapse, with the cell membrane pulling away from the cell wall and cytoplasmic content
leaking out of the cells. Buds, which did not withstand PVS2 treatment and storage in LN,
contained enlarged cells without any clear organization and differentiation. Only few of them
remained viable, while most of the cells lost their membrane integrity and their cytoplasmic
content was released in the intercellular zone (Fig. 3C). However, when apices withstood
storage in LN, they showed no severe tissue damage, except some cell disruptions, which did
not affect the normal development of the buds (Fig. 3D).
A B C
9. 107
Figure 3. Histological study of buds after PVS2 treatment and/or storage in LN. After 120-
min PVS2 treatment: (A) Bud showing normal development, (B) non viable bud, where
damaged cells release their cytoplasmic content to intracellular area (arrows). After 105-min
PVS2 treatment: (C) bud killed by cryopreservation, containing unorganized enlarged cells,
(D) cryopreserved bud with some cellular disruptions in the meristematic dome, which do not
impede regeneration. Note that in viable cells, sugars and intact nucleolus are stained, while
the dead cells remain sallow.
DISCUSSION
Long-term conservation of S. sempervirens via cryopreservation was achieved using
apical and basal buds excised from in vitro proliferating shoots. Pre-existing buds are
inherently genetically stable, and are thus considered ideal for long-term conservation of
clonally propagated species (25). However, these cultures are propagated in vitro and display
high metabolic activity because they are placed under optimal growth conditions. This makes
them highly hydrated and thus vulnerable to extensive desiccation and freezing injury during
cryopreservation (22). Such injury may be reduced or prevented by inducing freezing and
desiccation tolerance in cryopreserved samples. In the present study, the induction of
tolerance to LN storage was attempted by cold-hardening, pretreating apical and basal buds
with sucrose, and with PVS2 solution using three cryopreservation techniques: vitrification,
encapsulation-vitrification and droplet-vitrification. In these techniques, the exposure duration
to PVS2 of plant cells/tissues as well as the temperature at which the solution is applied are
regarded as the most critical factors affecting regrowth (9). However, it is well known that,
regardless of the technique selected, cryopreservation is a multi-step process, that includes
A B
C D
10. 108
preconditioning (e.g. cold-hardening), cryoprotection, storage in LN, rewarming and
regrowth, with each step optimized to ensure cryopreservation tolerance. The present study
focused on the optimization of the initial steps of a cryopreservation protocol, i.e.
preconditioning and preculture. Cold treatment is an excellent way of inducing cryotolerance,
however it is applicable only to cold tolerant species (9). For instance, results obtained in this
study showed that the highest survival of apical buds was achieved after cold-hardening. By
contrast, basal buds lost 50% of their regeneration ability immediately after this first
cryoprotective step but regained their regeneration ability (up to 86%) when they were
precultured on sucrose-enriched medium. Cold-hardening improves osmotic tolerance (5).
During sucrose preconditioning, cells are subjected to mild osmotic stress, which induces
metabolic changes and enhances chilling and desiccation tolerance. This is probably not only
due to cellular dehydration (5), but also to the activation of genes coding for factors, which
protect cells from cryopreservation-associated stress (12). In addition, absorbed sugars may
stabilize membranes by replacing water and forming hydrogen bonds with phospholipids (32).
This hypothesis is supported by our histological observations, which showed that only cells
that could accumulate sugars were able to maintain their viability, and that the amount of such
viable cells in tissues played a critical role in bud survival and regrowth.
Among the cryopreservation techniques tested, droplet-freezing was the only one
allowing post-rewarming regrowth. This may be related to the higher cooling rate achieved
with this protocol, due to the small volume of cryoprotectant solution in which explants are
frozen (only 3-4 µl vs. 200-500 µl in the other vitrification techniques). However, droplet-
freezing may also show some limitations. For instance, the small volume of the cryoprotectant
droplets employed to achieve such high cooling rates may lead to selecting smaller explants,
which, in turn, may have a lower regrowth potential. This was observed notably by Niino et
al. (19) with cherry shoot tips, where 3 mm shoot tips regenerated more easily and rapidly
after storage in LN compared with smaller ones (1-2 mm). Our observations showed that,
even though not all cells of buds, which did not withstand cryopreservation were dead, the
proportion of viable cells within the whole explants was a critical factor to determine survival
and regrowth. The amount of viable cells may be lower in smaller explants, thus rendering
their regrowth more difficult.
The composition of the culture medium used after storage in LN is of great importance
for regrowth. The medium employed should not only ensure rapid regrowth but also stimulate
survival of explants by reducing the concentration of toxic solutions used during the
cryopreservation protocol and by absorbing inhibitory substances and gases (such as ethylene)
which are induced by stress. Activated charcoal is a very effective medium additive for such
purposes (23). Its beneficial effect on in vitro regeneration of S. sempervirens has already
been reported (1, 3). In the present study, media containing activated charcoal proved to be
more suitable than other formulations tested, which were devoid of activated charcoal. This
positive effect was confirmed not only after storage in LN, but also following cold-hardening
and sucrose preculturing, treatments which are known to induce severe stress in the plant
material. Similarly to previous reports, also in our case the buds showed their regeneration
capacity by shoot sprouting and elongation when they were cultured on charcoal-containing
medium, while they immediately started to give rise to new buds upon their transfer back to
the original regeneration medium (MS1). This may be due to the fact that activated charcoal
adsorbs a large proportion of the growth regulators present in the culture medium, thus
modifying the quantity of growth regulators available to the plant material (32).
In our experiments, the highest regrowth achieved after LN storage was about 21.7%. It
is worth noting that the shoot cultures employed in this study had been maintained in vitro for
over 5 years by periodic 4-week subcultures. Ryynänen and Häggman (28) reported that silver
birch shoot tips excised from old cultures (over 55 months/60-67 subcultures) exhibited lower
11. 109
post-rewarming regrowth (14.8%) compared to (37.5%) younger cultures (20 months/20-25
subcultures). A similar detrimental effect of the age of cultures employed for cryopreservation
may have taken place in our experiments, which may partly explain the relatively low
regrowth percentages obtained after cryopreservation.
In conclusion, the present study, which represents the first application of
cryopreservation to in vitro shoot tips of a conifer species, demonstrated that S. sempervirens
shoots could be successfully cryopreserved using the droplet-vitrification technique. It is thus
possible to envisage that, in a not too distant future, cryopreservation will be employed to
ensure the safe and cost-effective long-term conservation of S. sempervirens germplasm.
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Accepted for publication10/10/2010
