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


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