The survival of in vitro shoot tips of Garcinia mangostana L. after cryopreservation by vitrification

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    The survival of in vitro shoot tips of Garcinia mangostana L.after cryopreservation by vitrification

    Sarah Ibrahim M. N. Normah

    Received: 15 July 2012 / Accepted: 14 February 2013 / Published online: 20 February 2013

    Springer Science+Business Media Dordrecht 2013

    Abstract This report highlights the first successful cryo-

    preservation protocol for shoot tips of Garcinia mangos-

    tana L. achieved by using vitrification technique. We

    investigated the effects of different temperatures and expo-

    sure periods to a plant vitrification solution 2 (PVS2), sucrose

    concentrations and preculture periods, and unloading treat-

    ments in steps of the vitrification protocol on the survival of

    G. mangostana shoot tips after cryopreservation. Exposure to

    PVS2 for 25 min gave beneficial effects with 10.4 1.8 %

    survival at 0 C with average water content of 1.1 0.3 g g-1 dry mass. Survival was 13.7 5.5 % when using

    preculture medium with full-strength Murashige and Skoog

    (MS) medium supplemented with 0.6 M sucrose for 2 days.

    A significant difference was observed in survival of shoot

    tips when treated with various sucrose concentrations in pre-

    culture which strengthens their importance towards enhanc-

    ing survival of shoot tips after cryopreservation. MS with

    0.4 M sucrose and 2 M glycerol applied as an unloading

    solution increased the survival of shoot tips to 44.1 6.5 %.

    Experiments on the effect of ascorbic acid were also con-

    ducted for each step of vitrification. Our results showed

    higher survival of 45.8 3.8 % but there were no signifi-

    cant effects compared with the control (without ascorbic

    acid). Further study on the recovery dark/light period was

    conducted. Survival of shoot tips significantly increased to

    50.0 16.7 % when subjected to 7 days in the dark before

    transferring to 16 h/8 h light/dark photoperiod. These stud-

    ies strengthen suggestions that cryopreservation through

    vitrification is possible for ex situ conservation of germ-

    plasm of this tropical recalcitrant species.

    Keywords Cryopreservation Garcinia mangostana Liquid nitrogen Shoot tips Ascorbic acid Vitrification


    Garcinia mangostana L., commonly known as mango-

    steen, is a major tropical fruit species belonging to the

    family Guttiferae (also known as Clusiaceae). There are

    many uses of mangosteen, and it is probably the most

    highly regarded tropical fruit tree. Its fruit is mostly eaten

    fresh, and both the rind and bark have several applications,

    as they possess anti-inflammatory, astringent, antibacterial,

    anti-tumour and anti-oxidative activities (Chairrungsri

    et al. 1996). Owing to these properties, various parts of the

    tree are used in Southeast Asia as traditional medicines for

    the treatment of abdominal pain, dysentery, wound infec-

    tions, suppuration and chronic ulcers.

    Mangosteen produces recalcitrant seeds that remain sen-

    sitive to desiccation both during development and after they

    are shed (Normah et al. 1995). Because conventional seed

    storage is not possible for recalcitrant seeds, the potentially

    safest method to conserve recalcitrant seeds for long periods

    without change is to store them in or above liquid nitrogen, a

    process known as cryopreservation (Berjak et al. 2000). At

    this temperature, biological deterioration is considered to be

    completely halted, and the germplasm can be stored for

    unlimited periods of time without modification including

    contamination (Engelmann 2011). Mangosteen seeds do not

    have differentiated embryos (Normah et al. 2011); thus, the

    S. Ibrahim

    School of Environmental and Natural Resource Sciences,

    Faculty of Science and Technology, University Kebangsaan

    Malaysia, 43600 Bangi, Selangor, Malaysia

    M. N. Normah (&)Institute of Systems Biology (INBIOSIS), Universiti

    Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia



    Plant Growth Regul (2013) 70:237246

    DOI 10.1007/s10725-013-9795-6

  • shoot tips are the ideal tissue to be used as explants for this

    important species. Cryopreservation of shoot tips offers

    long-term storage capability, maximum stability of the

    phenotypic and genotypic characteristics of the stored

    germplasm, and minimal storage space and maintenance

    requirements (Engelmann 1997).

    The vitrification technique is the most widely used cryo-

    preservation procedure, as it is easy to perform, is highly

    reproducible, and can be successfully applied to a wide range

    of tissues and plant species, especially those that are sensitive

    to dehydration and freezing (Reed and Uchendu 2008). Many

    studies have shown a high percentage of survival when the

    shoot tips were treated with Plant vitrification solution 2

    (PVS2) before plunging into liquid nitrogen (LN), including

    studies on apple and pear (Niino et al. 1997) and Populus

    alba (Lambardi et al. 2000) shoot tips.

    Optimising the time of exposure and the temperature during

    exposure to PVS2 are crucial aspects in the vitrification tech-

    nique. The duration of the diffusion of PVS2 through the cell

    membrane has to be long enough to ensure sufficient cell

    dehydration while avoiding cytotoxic effects. If cooled very

    rapidly, plant organs can be successfully cryopreserved at

    water contents as high as 1.11.6 g H2O g-1 dry mass (Wesley-

    Smith et al. 1992). Apart from that, using a stepwise method has

    proven to be more beneficial, especially with species that are

    relatively sensitive to dehydration (Takagi et al. 1997).

    Previous studies have also shown the importance of the

    preculture stage in many woody plant species. Xu et al.

    (2006) reported on shoot tips of Actinidia chinensis that the

    vacuoles became small and that the free water content in

    the cells decreased after preculture, indicating that the

    freezing tolerance and dehydration capacity increased with

    minimum damage while improving the resistance to cold

    and enabling the cell membrane to maintain a stable

    structure. Additionally, sucrose in preculture media acts as

    an osmoticum to accumulate sugar content inside cells and

    strengthen membrane integrity to withstand dehydration

    (Uragami et al. 1993). Many studies reported that

    increasing the sucrose concentration improved the survival

    percentage after cryopreservation including studies on

    shoot tips of Trichilia emetica (Varghese et al. 2009),

    Pyrus cordata (Chang and Reed 2001) and Mokara Golden

    Nugget Orchid (Safrinah et al. 2009).

    Storing plants at low temperatures could result in dele-

    terious effects caused by the chilling and freezing of the

    cells, which can then lead to an increased production of

    reactive oxygen species (ROS), often resulting in cell death

    (Day et al. 2000). ROS are believed to be produced during

    axis excision and in the steps of dehydration, exposure to

    and retrieval from cryogenic storage (Berjak et al. 2011).

    Antioxidants are believed to be able to arrest ROS before

    oxidative damage can occur and, thus, improved the sur-

    vival of blackberry shoot tips after cryopreservation

    (Uchendu et al. 2010a). This hypothesis is supported by the

    study of Chua and Normah (2011) in which ascorbic acid

    was proven beneficial for the survival of shoot tips of

    Nephelium ramboutan-ake after cryopreservation. In this

    study, the effects of the duration and temperature of PVS2

    exposure were evaluated, and the most suitable preculture

    medium and the best unloading solution for cryopreserva-

    tion were determined. The effect of ascorbic acid in each

    step of the vitrification procedure was also examined.

    In addition, dark/light period controls explant survival

    and regrowth. Touchell and Walters (2000) reported that

    recovery percentages increased when cultures were ini-

    tially maintained in the dark as compared to light. This is

    suggested to be because damaging consequences of photo-

    oxidation would be avoided or minimized in cultures

    maintained in dark or minimal light conditions (Withers

    1988). Thus, further research was conducted to investigate

    the potential effects of dark/light exposure during recovery

    of the cryopreserved shoot tips.

    Materials and methods

    Plant materials and culture conditions

    The fruits of G. mangostana were obtained from Puchong,

    Selangor, Malaysia, in the months of July and August of

    2010. The fleshy edible parts were peeled off the seeds, and

    the rubbery testa enclosing the seeds was removed using

    forceps and a blade. The seeds were then washed under

    running water for 20 min before decontamination with

    80 % alcohol for 12 min. The seeds were then disinfected

    using 20 % Clorox (5.25 % sodium hypochlorite; The

    Clorox Company, USA) with two drops of Tween 20 for

    20 min. The seeds were then rinsed three times with sterile

    distilled water, blotted dry with sterile filter paper and

    placed in a Petri dish before culturing on Murashige and

    Skoog (MS) medium (1962) supplemented with 4 mg l-1

    benzylaminopurine (BAP) and 2.5 g l-1 gelrite. Seeds

    were cut into three segments to maximise the shoot

    induction area (Fig. 1). The explants were then subcultured

    onto the same medium every 3 weeks. The explants were

    subcultured for a week before the shoot tips of approxi-

    mately 12 mm long were used for cryopreservation. All

    cultures were incubated under fluorescent light at

    23 2 C, with a light intensity of 22.26 lE cm-2 s-1

    (3,000 lux) and 16 h/8 h light/dark photoperiod.

    Cryopreservation procedures

    Cryopreservation of the shoot tips was carried out according

    to the method of Sakai et al. (1990), with slight modifica-

    tions. Excised shoot tips were precultured on MS medium

    238 Plant Growth Regul (2013) 70:237246


  • supplemented with 0.3 M sucrose and 5 % DMSO for 48 h.

    The shoot tips were then transferred to 1.8 ml cryovials and

    treated with 1 ml of loading solution (2 M glycerol ?

    0.4 M sucrose) for 20 min at 25 C. The loading solutionwas then removed, and 1 ml of PVS2 solution (30 %

    glycerol, 15 % ethylene glycol, 15 % DMSO and 0.4 M

    sucrose) (Sakai et al. 1990) was added followed by incu-

    bation for different times at 25 C. The PVS2 solution wasthen removed, and the shoot tips were immediately trans-

    ferred to a new cryovials to which 0.5 ml of fresh PVS2

    solution was added before the vials were rapidly plunged

    into liquid nitrogen (LN) (-196 C) for at least 1 h. Thecryovials containing the frozen shoot tips were then rapidly

    thawed in a sterile-water bath at 38 2 C for 4045 swith constant agitation, and the shoot tips were rinsed with

    unloading solution (MS containing 0.4 M sucrose and 2 M

    glycerol) at 25 C three times (*5 min) before culturing onrecovery medium. The control, which was not immersed in

    LN, was directly rinsed with unloading solution at 25 Cthree times (*5 min) and then cultured on recovery med-ium. The recovery medium consisted of MS supplemented

    with 4 mg l-1 BAP and 2.5 g l-1 gelrite. The cultures were

    incubated under fluorescent light at 23 2 C, with a lightintensity of 22.26 lE cm-2 s-1 (3,000 lux) and subculturedevery week.

    Effects of the plant vitrification solution (PVS2)

    exposure procedure on the survival of the shoot tips

    after cryopreservation

    A preliminary study was conducted to study the sensitivity

    of shoot tips to PVS2 exposure. Excised shoot tips were

    precultured on MS medium supplemented with 0.3 M

    sucrose and 5 % DMSO for 48 h. Shoot tips were then

    exposed to 100 % PVS2 for 0, 5, 10, 15, 20, 25, 30, 35 and

    40 min. Treated shoot tips were then directly cultured on

    the recovery medium.

    The best exposure period for survival of shoot tips was

    used for further study on the effects of different concen-

    trations of PVS2 at different periods of exposure. The shoot

    tips were exposed to seven treatments at 25 C; directexposure for 0, 5, 10, 15, 20 and 25 min in 100 % PVS2;

    and step-wise approach for 15 min in 50 % PVS2 followed

    by 10 min in 100 % PVS2. With the results of this

    experiment, the effects of two different temperatures (0 and

    25 C) of PVS2 exposure on shoot tips were conducted.

    Effects of preculture on the survival of the shoot tips

    after cryopreservation

    Studies on the preculture stage were conducted to improve

    survival after cryopreservation. The effects of different

    concentrations of sucrose (0, 0.3, 0.6 and 0.9 M) and the

    exposure period (1 and 2 days) in the culture medium were

    studied. DMSO at 5 % was added to all preculture media.

    After obtaining the highest survival from this experiment,

    the effects of two strengths of MS media (full and half

    strengths) were then studied. The PVS2 exposure period

    and temperature and sucrose concentration for preculture

    were based on the above experiments.

    Effects of unloading solution on the survival

    of the shoot tips after cryopreservation

    Two types of unloading solutions, liquid MS supplemented

    with 1.2 M sucrose (Sakai et al. 1990) and MS supple-

    mented with 0.4 M sucrose and 2 M glycerol (suggested by

    Matsumoto et al. 1998 for tropical species), were evaluated

    for their effect on the survival of the explants. The PVS2

    exposure period and temperature, sucrose concentration

    and MS medium strength for preculture were based on the

    above experiments.

    Effects of ascorbic acid on the survival of the shoot tips

    after cryopreservation

    Effects of ascorbic acid on each step of vitrification (pre-

    culture, loading, PVS2, unloading and recovery) protocol

    were studied. Ascorbic acid at 0.28 mM was added in the

    vitrification solutions to study its effects on the survival of

    the shoot tips after cryopreservation. The sucrose concen-

    tration, MS medium strength for preculture, PVS2 expo-

    sure period and temperature and the unloading solution

    were ultimately selected based on the above experiments.

    Effects of the dark/light period during recovery

    on the survival of the shoot tips after cryopreservation

    The effects of dark/light period during recovery on the sur-

    vival of shoot tips were tested. After exposed to unloading

    0.5 cm

    (a) (b) (c)Fig. 1 Seeds of G. mangostana: a Seed with testa b Seed withouttesta c Seed that has been cut into three segments

    Plant Growth Regul (2013) 70:237246 239


  • solution, explants were cultured on the recovery medium (as

    described above) and placed under dark conditions for 0, 3,

    5, and 7 days before transferring to 16 h/8 h light/dark

    photoperiod. All steps and procedures for cryopreservation

    were based on the experiments described above.

    Statistical analysis

    All of the data obtained from the experiments were statisti-

    cally analysed using SAS software. Data in percentiles were

    arcsin transformed before subjecting them to a parametric

    statistical test. The treatment means were compared using

    Duncans Multiple Range Test (DMRT) and standard

    deviation (SD). Fifteen shoot tips were used for each treat-

    ment, and each treatment was replicated three times. The

    survival of the shoot tips were observed and recorded every

    3 days. The shoot tips were considered to have survived if

    they remained green and showed indication of growth,

    whereas those that turned brown were considered dead.


    Effects of the plant vitrification solution (PVS2)

    exposure procedure on the survival of the shoot tips

    after cryopreser...


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