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CryoLetters 32 (2), 99-110 (2011)© CryoLetters, businessoffice@cryoletters.org

CRYOPRESERVATION OF REDWOOD (SEQUOIA SEMPERVIRENS(D. DON.) ENDL.) IN VITRO BUDS USING VITRIFICATION-BASED

TECHNIQUES

E. A. Ozudogru1*, E. Kirdok1, E. Kaya1, M. Capuana2, C. Benelli3 and F. Engelmann4, 5

1Gebze Institute of Technology, Faculty of Science, Department of Molecular Biology andGenetics, Istanbul Caddesi, No 101, 41400, Gebze (Kocaeli), Turkey 2Istituto di GeneticaVegetale, CNR/Consiglio Nazionale delle Ricerche, Polo Scientifico, via Madonna del Piano,50019 Sesto Fiorentino (Firenze), Italy.3Istituto per la Valorizzazione del Legno e delle Specie Arboree, CNR/Consiglio Nazionaledelle Ricerche, Polo Scientifico, via Madonna del Piano, 50019 Sesto Fiorentino (Firenze),Italy.4Institut de Recherche pour le Développement (IRD), UMR DIAPC, 911 avenue Agropolis,BP 64501, 34394 Montpellier cedex 5, France.5Bioversity International, Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome,Italy.*Corresponding author email: elif@gyte.edu.tr or aylin_ozudogru@yahoo.com

Abstract

In this study, the efficiency of three vitrification-based cryopreservation techniques, i.e.vitrification, encapsulation-vitrification and droplet-vitrification were compared forcryopreserving Sequoia sempervirens apical and basal buds sampled from in vitro shootcultures. The effect of cold-hardening of mother-plants and of bud culture medium andsucrose preculture was also investigated. Culture of apical and basal buds sampled from cold-hardened mother-plants on Quoirin and Lepoivre medium with activated charcoal had apositive effect on regrowth. Only droplet-vitrification ensured survival and regrowth aftercryopreservation. After cryopreservation, regeneration of apical buds was possible for PVS2exposure durations between 90 and 180 min but it remained low, with a maximum of 18%after 135 min treatment. With basal buds, regeneration after cryopreservation was possibleover a larger range of PVS2 treatment durations, between 30 and 180 min. The highestregeneration percentage was slightly higher (22%) than that measured with apical buds, andwas also achieved after 135 min PVS2 exposure.Keywords: conservation, genetic resources, redwood, cryopreservation, droplet-vitrification.

INTRODUCTION

Sequoia sempervirens (D. Don) Endl., also called ‘redwood’ due to the reddish browncolour of its heart-wood (1), is an evergreen conifer tree which originates from the Pacificcoast of the USA and which has been introduced to various European countries. Redwood is

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highly valuable for ornamental purposes, reforestation, timber and plywood production.However, its conservation is problematic due to its low rooting capacity, shoot dormancy, lowseed germinability (about 10%, 2) and low seedling viability (4, 7, 15).

In vitro techniques are widely used for multiplication and conservation of species whosepropagation 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 invitro 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 ofplant species, originating both from tropical and temperate climates. Among the sevenvitrification-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 theproduction of artificial seeds (6). Explants are encapsulated in alginate beads, pregrown inliquid medium enriched with sucrose for 1 to 7 days, partially desiccated in the air current of alaminar 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 andsomatic embryos of numerous species (11). Vitrification involves treatment of samples with aloading solution containing cryoprotectants at intermediate concentration, usually 2 Mglycerol + 0.4 M sucrose (17), dehydration with highly concentrated vitrification solutions,usually the PVS2 (30) and PVS3 solutions, (20), rapid cooling and rewarming, removal ofcryoprotectants in an unloading solution and regrowth. This procedure was developed forapices, cell suspensions and somatic embryos of numerous species (29, 31). Encapsulation-vitrification is a combination of encapsulation-dehydration and vitrification procedures, wheresamples are encapsulated in alginate beads, then subjected to vitrification solutions. It hasbeen applied to apices of an increasing number of species (31). The droplet-vitrificationtechnique 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 inminute droplets of vitrification solution and cooled rapidly in liquid nitrogen (LN). The mainadvantage of this technique is the very high cooling and warming rates achieved, thanks to thesmall volume of cryoprotectant solution employed and to the fact that explants are plungeddirectly in liquid nitrogen for cooling and in unloading medium for rewarming. This avoidsthe buffering effect of the cryotube and of the relatively large volume of cryoprotectantsolutions employed in other vitrification protocols.

This study aimed at determining a cryopreservation protocol for apical and basal budssampled from S. sempervirens in vitro shoot cultures. Three techniques were compared:vitrification, encapsulation-vitrification and droplet-vitrification. Histo-cytologicalobservations were performed to observe the effect of the successive steps of thecryopreservation protocol on the structural integrity of the buds.

MATERIALS AND METHODS

Plant materialIn vitro shoot cultures of S. sempervirens (D. Don.) Endl. were initiated from an old

elite tree, growing in a public garden of Florence (Italy).

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Standard culture conditionsCultures 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 wasadjusted to 5.8 with 1N NaOH or HCl before addition of agar and the medium was autoclavedfor 20 min at 121°C. Cultures were incubated at 23±2°C under a 16 h light/8 h darkphotoperiod, with a light intensity of 36.3 µmol m-2 s-1 provided by cool daylight fluorescentlamps.

Preliminary experimentsPrior 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 andbasal buds.

Cold-hardening of in vitro shoot culturesShoot 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 MSmedium with 20 g l-1 activated charcoal; (iv) QL0: hormone-free Quoirin and Lepoivremedium (QL, 24); (iv) QL1: QL medium with 1 mg l-1 BA; and (vi) QL + AC: hormone-freeQL medium with 20 g l-1 activated charcoal. Survival and regrowth of buds were recordedafter 4 weeks.

Sucrose preculture of apical and basal budsFollowing 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, andcultured for 24, 48 or 72 h under standard conditions. They were then transferred to QL + ACmedium and subcultured at 4 week intervals. Regrowth of buds was recorded at the end of thefirst subculture period.

Cryopreservation of apical and basal buds using vitrification-based techniquesThree 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 weresampled 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 (apicalbuds) or for 72 h on QL medium with 0.25 M sucrose (basal buds), then treated as describedbelow.

PVS2 vitrificationApical 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 MSmedium; 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 Msucrose, 30) and buds were treated with PVS2 for up to 180 min at 0°C. Following the PVS2treatment, half of the apical and basal buds were suspended in 0.6 ml fresh PVS2 and directlyimmersed in LN, while the other half (controls) were washed in unloading solution (liquid MSmedium with 1.2 M sucrose, 30) for 20 min at 25°C, then plated on QL + AC medium under

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standard conditions for regrowth. At the end of the first 4-week subculture, buds weretransferred to MS1 medium for induction of multiple buds/shoots. After at least 1 h storage inLN, cryopreserved samples were rewarmed in a water-bath at 40°C for 2 min, then treated ascontrol samples.

Encapsulation-vitrificationApical 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 completepolymerization of calcium alginate, collected on a sterile sieve and washed with steriledistilled 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 treatedwith 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-vitrificationSterile 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 weredropped 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 thebuds (controls) were immersed in unloading solution for 20 min and plated on QL + ACmedium for regrowth. The other half of the explants, the aluminium foils were plunged in 2ml Nalgene® cryovials previously filled with LN, which were then transferred to LN tanks forstorage. Rewarming was performed at room temperature by retrieving the aluminium foilsfrom LN and immersing them in the unloading solution for 20 min at 25°C. Buds were thentransferred on QL + AC medium and incubated under standard culture conditions forregrowth.

Histological observationsApical 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 pH7.2) at 4°C for 24 h. They were dehydrated for 24 h at 4°C in ethylene glycol monomethylether, then in absolute ethanol and embedded in LKB historesin according to the procedure ofYeung 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 Black10B (27).

Data collection and statistical analysisTwo replicates of 30 apical and basal buds were used for each treatment of

cryopreservation experiments and all experiments were repeated at least three times. Onlybuds not showing any symptom of contamination were considered for data evaluation.Regrowth (% of buds which had elongated and/or from which new buds developed) wasevaluated 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 bythe least significant difference (LSD) test at P ≤ 0.05 to compare means.

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RESULTS

Effect of shoot culture cold-hardening on regrowth of apical and basal budsTable 1 presents the results of the ANOVA performed on the effect of bud type,

medium composition and cold-hardening on S. sempervirens regrowth. All interactionsstudied 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 compositionand 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 indicatesignificant 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 typeApical budsBasal buds

33.9 A16.0 A

Culture mediumMS0MS1MS + ACQL0QL1QL + AC

36.2 B5.2 D

47.6 B18.0 C

0.2 E68.8 A

Cold-hardening0 (control)1 week2 weeks3 weeks4 weeks

50.1 A21.4 B14.1 B21.3 B19.0 B

* Percentage data were subjected to arcsin % transformation before analysis by ANOVA,followed by LSD test at P ≤ 0.01.

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The bud type (apical or basal) employed had no effect on regrowth (Table 2). Additionof BA in the culture medium was detrimental to regrowth with both mineral solutions. Whenno growth regulators were present in the medium, regrowth was intermediate. Addition ofactivated charcoal in QL medium led to highest regrowth. Cold-hardening always reducedregrowth, whatever the duration of cold-hardening treatment employed.

Effect of sucrose preculture on regrowth of apical and basal budsAll 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 andpreculture 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 onregrowth (%) of S. sempervirens buds. For each treatment, different letters indicatesignificant differences at P≤0.01.

Treatment Regrowth (%)*

Bud typeApical budsBasal buds

76.5 A61.4 B

Sucrose preculture duration24h48h72h

63.9 A62.6 A63.5 A

Sucrose concentration0.12M0.25M0.50M

83.1 A78.7 A26.1 B

* Percentage data were subjected to arcsin % transformation before analysis by ANOVA,followed by LSD test at P ≤ 0.01.

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Table 4 shows the effect of bud type, sucrose preculture duration and sucroseconcentration on regrowth of S. sempervirens buds. Apical buds had a significantly higherregrowth than basal ones. Sucrose preculture duration had no significant effect on regrowth,while lower sucrose concentrations induced higher regrowth.

Cryopreservation of apical and basal budsAmong the three techniques tested, vitrification, encapsulation-vitrification and droplet-vitrification, only the latter resulted in survival and regrowth after cryopreservation under theexperimental 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 PVS2exposure durations comprised between 90 and 180 min but it remained low, with a maximumof 18% after 135 min treatment.

Figure 1. Response of apical (A) and basal buds (B) to PVS2 treatment (control) or to PVS2treatment and storage in LN (LN +) for droplet-vitrification. In A and B, droplet-vitrificationwas applied after a 4 week cold-hardening and 48 h preculture with 0.12 M sucrose (apicalbuds) or 72 h preculture with 0.25 M sucrose (basal buds).

A A

B BB B

BBCBC

Ca

aa ab bb C C

ab

aa

aabbb

abb

A

AA

A A AAB

B BB B

B

A

B

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Figure 2. Cryopreservation of Sequoia sempervirens. Shoot tips, placed inside PVS2 dropson aluminium foils (A, bar, 2 cm). Regrowth of an apical (B, bar, 1 cm) and basal (C, bar, 1cm) bud after PVS2 treatment and exposure to LN.

With basal buds, regrowth of control explants also decreased regularly, but moreslowly compared with apical buds, reaching 18% after 180 min PVS2 exposure (Fig. 2B).After cryopreservation, regrowth was possible over a larger range of PVS2 treatmentdurations, 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 PVS2exposure.

Histological observationsS. 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 budswere able to maintain their integrity (Fig. 3A). These viable cells (especially in the four-fiveexternal layers of the meristematic dome) had a dense cytoplasm, small vacuoles, an intactnucleolus, 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 notwithstand the PVS2 treatment, appeared sallow. Similarly, almost all the cells of buds, whichdid not withstand PVS2 treatment, were relatively large (Fig. 3B). These cells tended to swelland collapse, with the cell membrane pulling away from the cell wall and cytoplasmic contentleaking 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 themremained viable, while most of the cells lost their membrane integrity and their cytoplasmiccontent was released in the intercellular zone (Fig. 3C). However, when apices withstoodstorage in LN, they showed no severe tissue damage, except some cell disruptions, which didnot affect the normal development of the buds (Fig. 3D).

A B C

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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, wheredamaged cells release their cytoplasmic content to intracellular area (arrows). After 105-minPVS2 treatment: (C) bud killed by cryopreservation, containing unorganized enlarged cells,(D) cryopreserved bud with some cellular disruptions in the meristematic dome, which do notimpede regeneration. Note that in viable cells, sugars and intact nucleolus are stained, whilethe dead cells remain sallow.

DISCUSSION

Long-term conservation of S. sempervirens via cryopreservation was achieved usingapical and basal buds excised from in vitro proliferating shoots. Pre-existing buds areinherently genetically stable, and are thus considered ideal for long-term conservation ofclonally propagated species (25). However, these cultures are propagated in vitro and displayhigh metabolic activity because they are placed under optimal growth conditions. This makesthem highly hydrated and thus vulnerable to extensive desiccation and freezing injury duringcryopreservation (22). Such injury may be reduced or prevented by inducing freezing anddesiccation tolerance in cryopreserved samples. In the present study, the induction oftolerance to LN storage was attempted by cold-hardening, pretreating apical and basal budswith sucrose, and with PVS2 solution using three cryopreservation techniques: vitrification,encapsulation-vitrification and droplet-vitrification. In these techniques, the exposure durationto PVS2 of plant cells/tissues as well as the temperature at which the solution is applied areregarded 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

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preconditioning (e.g. cold-hardening), cryoprotection, storage in LN, rewarming andregrowth, with each step optimized to ensure cryopreservation tolerance. The present studyfocused 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 thisstudy showed that the highest survival of apical buds was achieved after cold-hardening. Bycontrast, basal buds lost 50% of their regeneration ability immediately after this firstcryoprotective step but regained their regeneration ability (up to 86%) when they wereprecultured on sucrose-enriched medium. Cold-hardening improves osmotic tolerance (5).During sucrose preconditioning, cells are subjected to mild osmotic stress, which inducesmetabolic changes and enhances chilling and desiccation tolerance. This is probably not onlydue to cellular dehydration (5), but also to the activation of genes coding for factors, whichprotect cells from cryopreservation-associated stress (12). In addition, absorbed sugars maystabilize membranes by replacing water and forming hydrogen bonds with phospholipids (32).This hypothesis is supported by our histological observations, which showed that only cellsthat could accumulate sugars were able to maintain their viability, and that the amount of suchviable cells in tissues played a critical role in bud survival and regrowth.

Among the cryopreservation techniques tested, droplet-freezing was the only oneallowing post-rewarming regrowth. This may be related to the higher cooling rate achievedwith this protocol, due to the small volume of cryoprotectant solution in which explants arefrozen (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 cryoprotectantdroplets 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 etal. (19) with cherry shoot tips, where 3 mm shoot tips regenerated more easily and rapidlyafter 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, theproportion of viable cells within the whole explants was a critical factor to determine survivaland regrowth. The amount of viable cells may be lower in smaller explants, thus renderingtheir regrowth more difficult.

The composition of the culture medium used after storage in LN is of great importancefor regrowth. The medium employed should not only ensure rapid regrowth but also stimulatesurvival of explants by reducing the concentration of toxic solutions used during thecryopreservation 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 suchpurposes (23). Its beneficial effect on in vitro regeneration of S. sempervirens has alreadybeen reported (1, 3). In the present study, media containing activated charcoal proved to bemore suitable than other formulations tested, which were devoid of activated charcoal. Thispositive effect was confirmed not only after storage in LN, but also following cold-hardeningand sucrose preculturing, treatments which are known to induce severe stress in the plantmaterial. Similarly to previous reports, also in our case the buds showed their regenerationcapacity by shoot sprouting and elongation when they were cultured on charcoal-containingmedium, while they immediately started to give rise to new buds upon their transfer back tothe original regeneration medium (MS1). This may be due to the fact that activated charcoaladsorbs a large proportion of the growth regulators present in the culture medium, thusmodifying 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%. Itis worth noting that the shoot cultures employed in this study had been maintained in vitro forover 5 years by periodic 4-week subcultures. Ryynänen and Häggman (28) reported that silverbirch shoot tips excised from old cultures (over 55 months/60-67 subcultures) exhibited lower

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post-rewarming regrowth (14.8%) compared to (37.5%) younger cultures (20 months/20-25subcultures). A similar detrimental effect of the age of cultures employed for cryopreservationmay have taken place in our experiments, which may partly explain the relatively lowregrowth percentages obtained after cryopreservation.

In conclusion, the present study, which represents the first application ofcryopreservation to in vitro shoot tips of a conifer species, demonstrated that S. sempervirensshoots could be successfully cryopreserved using the droplet-vitrification technique. It is thuspossible to envisage that, in a not too distant future, cryopreservation will be employed toensure the safe and cost-effective long-term conservation of S. sempervirens germplasm.

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Accepted for publication10/10/2010