Cryopreservation of in vitro-grown shoot tips of Alnus glutinosa (L.) Gaertn.

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    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 (0.51 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 mixtureof 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 thenplaced in 100 % PVS2, for 30 min at 0 C. The solutionwas 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 thenwashed twice, for 10 min at 25 C, with 1.2 M sucrosesolution, 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


    Alnus glutinosa is a medium-sized tree, of height 1722 m

    and diameter 6070 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. CorredoiraInstituto de Investigaciones Agrobiologicas de Galicia, CSIC,

    Apartado 122, 15705 Santiago de Compostela, Spain


    L. V. Janeiro

    INLUDES, Diputacion Provincial de Lugo, Ronda de la Muralla

    140, 27004 Lugo, Spain


    Acta Physiol Plant (2014) 36:109116

    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 2530-

    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 5060 lmol m-2 s-1, provided bycool-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 (0.51 mm in

    length 9 0.50.8 mm in width, and consisted of the apical

    meristem and 26 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 theshoot tips were washed twice with unloading solution

    110 Acta Physiol Plant (2014) 36:109116


  • (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 dividedinto 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 othergroup 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.


    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 shoottips 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 weredehydrated with 100 % PVS2 for 30, 60 or 90 min at 0 C.LN exposure, rinsing and recovery were as described


    The effect of the exposure temperature (0 or 25 C) wasstudied 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 (2530 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 0.81, 11.5 and 1.52 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 23, 46 and 78 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 2025 years, and clone R1 was taken from the

    crown of a tree aged 2530 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 1012

    shoot tips per dish (3036 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) 36:109116 111


  • experimental factors was analyzed by a Students 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.


    Cryopreserved alder shoot tips turned blackish brown

    within 1 day of thawing, but surviving shoot tips turned

    green within 1012 days on recovery medium, and suc-

    cessfully recovered shoot tips resumed growth within

    23 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 23 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.


    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 0.20.3 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 indarkness 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 besttreatment for cryopreservation of A. glutinosa shoot tips by


    Shoot tips exposed to PVS2 at 25 C (50 % PVS2 for30 min plus 100 % PVS2 for 30 min) did not survive.

    Fig. 1 Shoots developing from successfully cryopreserved shoot tipsof A. glutinosa (clone R4) 10 weeks after plating in recovery medium











    Control 0,2 M 2 d 0,2 M 3d 0,3 M 2d 0.3 M 3d




    Sucrose concentration (M) and preculture duration (days)



    a a



    a a









    Fig. 2 Effect of preculture period and sucrose concentration onsurvival 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) 36:109116


  • 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 0.51 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).


    We report here a protocol for the cryopreservation of alder

    shoot tips by vitrification. Shoot tips are widely used in











    30 min 60 min 90 min 15+15 min 30+30 min 45+45 min




    100% PVS2 50% PVS2 + 100% PVS2











    Fig. 3 Survival and recovery ofvitrified alder shoot tips cooled

    to -196 C by two-stepvitrification 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











    SCC 1w dark + 1w dim light








    Fig. 4 Effect of the culture conditions during the recovery ofcryopreserved 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











    3 w 6w 9w




    Age of the donor of the shoot tips (weeks)






    b b

    Fig. 5 Effect of age of donor plants on survival and recovery ofcryopreserved 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) 36:109116 113


  • 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 vitrificationthan 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 resultswere significantly improved (by more than 40 %) by

    application of the two-step vitrification procedure for

    6090 min at 0 C. The two-step vitrification procedurehas 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 by100 % PVS2 for 10 min at 25 C yielded the highest rateof 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. Kaityet al. (2008) reported a 6173 % 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 7984 % for shoot tips of

    Emmenopterys henryi, which were dehydrated with 60 %

    PVS2 solution for 30 min at 0 C and then 100 % PVS2for 40 min at 0 C. Careful control of the dehydration











    0.5 mm 1 mm 2 mm




    Shoot tips size








    Fig. 6 Effect of shoot tip size (0.52 mm) on survival and recoveryof 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











    Clone G1 Clone R1 Clone R4





    ab a




    Fig. 7 Effect of genotype on survival and recovery of aldercryopreserved 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) 36:109116


  • procedure appears to be essential for the successful cryo-

    preservation of plant tissues by vitrification (Panis et al.


    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 0.51 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 (23-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 (Garca 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 usefuladvice and suggestions and Carlos Suarez for technical assistance.

    This study was funded by INLUDES (Diputacion Provincial de



    Arias Padro MD, Fratarelli A, Sgueglia A, Condello E, Damiano C,

    Caboni E (2012) Cryopreservation of white mulberry (Morus

    alba L.) by encapsulation-dehydration and vitrification. Plant

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    Cryopreservation of in vitro-grown shoot tips of Alnus glutinosa (L.) Gaertn.AbstractIntroductionMaterials and methodsPlant material and culture conditionsCryopreservation proceduresEffect of loading and vitrification solutionsPrecultureEfficiency of the two-step PVS2 exposureEffect of the culture conditions during recoveryEffects of age of shoot tip donorsEffect of the size of the shoot tipsEffect of genotype

    Statistical analysis

    ResultsEffect of loading and PVS2 solutionPrecultureTwo-step vitrification procedureEffect of the culture conditions during recoveryEffects of age of donor plants on survivalEffect of the size of the shoot tipsEffect of genotype



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