Cryopreservation of sour orange (Citrus aurantium L.) shoot tips

Embed Size (px)

Text of Cryopreservation of sour orange (Citrus aurantium L.) shoot tips



    Department of Plant Production, Faculty of Agriculture, Jordan University of Science and Technology, P.O. Box 3030, Irbid, Jordan

    (Received 23 November 2002; accepted 5 July 2002; editor S. A. Merkle)


    The objective of this study was to establish a cryopreservation protocol for sour orange (Citrus aurantium L.).

    Cryopreservation was carried out via encapsulationdehydration, vitrification, and encapsulationvitrification on shoot

    tips excised from in vitro cultures. Results indicated that a maximum of 83% survival and 47% regrowth of encapsulated

    dehydrated and cryopreserved shoot tips was obtained with 0.5 M sucrose in the preculture medium and further

    dehydration for 6 h to attain 18% moisture content. Dehydration of encapsulated shoot tips with silica gel for 2 h resulted

    in 93% survival but only 37% regrowth of cryopreserved shoot tips. After preculturing with 0.5 M sucrose, 80% of the

    vitrified cryopreserved shoots survived when 2 M sucrose plus 10% dimethyl sulfoxide (DMSO) was used as a

    cryoprotectant for 20 min at 258C. Survival and regrowth of vitrified cryopreserved shoot tips were 67% and 43%,respectively, when 0.4 M sucrose plus 2 M glycerol was used as a loading solution followed by application of 100% plant

    vitrification solution (PVS2) for 20 min. Increased duration of exposure to the loading solution up to 60 min increased

    survival (83%) and regrowth (47%) of cryopreserved shoot tips. With encapsulationvitrification, dehydration with 100%

    PVS2 for 2 or 3 h at 08C resulted in 50 or 57% survival and 30 or 40% regrowth, respectively, of cryopreserved shoot tips.

    Key words: preservation; cryopreservation; encapsulation; vitrification.


    Cryopreservation in liquid nitrogen (21968C) is a preservation

    method in which cell division and metabolic and biochemical

    processes are arrested (Niino and Sakai, 1992). Thus, the plant

    material is stored without deterioration or modification for an

    unlimited time (Lambardi et al., 2000) and genetic stability and

    regeneration potential of the cryopreserved material are

    maintained (Rajasekaran, 1996). Cryopreservation may be

    achieved through encapsulationdehydration, vitrification, or


    With encapsulationdehydration, explants are encapsulated in

    beads, dehydrated, and then cooled rapidly in liquid nitrogen

    (Bachiri et al., 1995; Niino et al., 1995; Shibli et al., 1999; Sakai

    et al., 2000). This method is simple, inexpensive and the high

    genetic stability of the cryopreserved material can be maintained

    (Kartha and Engelmann, 1994).

    In vitrification, tissues are dehydrated with a highly concentrated

    osmoticum to avoid ice formation during cryopreservation and

    thawing (Bachiri et al., 1995). This technique is simple, does not

    need expensive cooling apparatus, and can be applied to a wide

    range of plant material (Niino and Sakai, 1992; Matsumoto et al.,

    1994). The technique consists of three major phases (Engelmann,

    1997; Tahtamouni and Shibli, 1999). The loading phase involves

    treatment of tissue with cryoprotectants or diluted vitrification

    solutions (Ashmore, 1997). The dehydration phase involves

    dehydrating plant tissue with a highly concentrated vitrification

    solution (Sakai et al., 1991). The plant vitrification solution is an

    aqueous cryoprotectant solution in which living systems can be

    cooled slowly without appreciable intra- or extracellular ice

    formation (Fahy et al., 1987). This solution increases the osmotic

    potential of the external medium (Reed, 1995), resulting in flow of

    water out of the cells and dehydration of tissue (Ashmore, 1997). A

    single cryoprotectant, usually dimethyl sulfoxide (DMSO), is

    effective (Goldner et al., 1991) although a cryoprotectant mixture

    may be more effective for some plant species (Sakai et al., 1990).

    High concentrations of cryoprotectants in the medium lead to

    reduced survival due to their toxic effect (Reed, 1995). The duration

    of contact between the explant and the vitrification solution is a

    critical parameter affecting the survival percentage of the

    cryopreserved plant material (Engelmann, 1997). The dehydration

    period generally increases with the size of the explant used

    (Ashmore, 1997). Permeating the dehydration step at 08C reducesthe vitrification solution toxicity (Ashmore, 1997), thus broadening

    duration of exposure to the vitrification solution and increasing

    survival percentage of the cryopreserved plant tissues (Engelmann,

    1997). The unloading phase starts after rapid warming, where plant

    vitrification solution (PVS2) is drained out of the cryotubes and

    replaced with 1.2 M sucrose for 1030 min at 258C (Ashmore,


    Encapsulationvitrification, which is a combination of encapsu-

    lation and vitrification (Engelmann, 1997), reduces the injury effect

    of the vitrification solution on explants (Ashmore, 1997) and results

    in higher survival rates (Hirai and Sakai, 1999). With this

    technique, the plant material is osmoprotected with a mixture*Author to whom correspondence should be addressed: Email shibli@just.

    In Vitro Cell. Dev. Biol.Plant 38:602607, NovemberDecember 2002 DOI: 10.1079/IVP2002349q 2002 Society for In Vitro Biology1054-5476/02 $10.00+0.00


  • containing 2 M glycerol and 0.4 M sucrose during the encapsulation

    process and then hydrated with PVS2 for 23 h (Matsumoto et al.,

    1995; Hirai and Sakai, 1999; Sakai et al., 2000). Exposing

    encapsulated shoot tips to the vitrification solution at 08C is neededto reduce the injurious effect of the vitrification solution and thus

    the time that cells are exposed to osmotic stress may be extended

    (Hirai and Sakai, 1999). With this technique, dehydration and

    freezing tolerance is achieved through capsules osmoprotected with

    vitrification solutions (Hirai and Sakai, 1999; Shibli and

    Al-Juboory, 2000). This technique is easy to handle, saves the

    time needed for dehydration (Hirai et al., 1998; Sakai et al., 2000;

    Shibli and Al-Juboory, 2000), and results in growth recovery which

    is much earlier than that with encapsulationdehydration

    (Matsumoto et al., 1995; Hirai and Sakai, 1999). Encapsulation

    vitrification has been described as a cryogenic protocol with high

    potential for large-scale cryopreservation (Hirai et al., 1998; Shibli

    and Al-Juboory, 2000).

    Sour orange (Citrus aurantium L.) is used as a rootstock and has

    several advantages over the commercially used seedling rootstocks,

    including resistance to several viral diseases and improvement of

    fruit quality of the grafted species (Samson, 1986). Sour orange is

    endangered and the use of other rootstocks will result in a decline in

    performance of sour orange rootstock with time due to unfavorable

    environmental conditions, especially drought and salinity. This

    mandates preservation of valuable genetic resources of sour orange

    for future use and improvement. Therefore, this study was initiated

    to develop protocols for cryopreservation of sour orange via

    encapsulationdehydration, vitrification, and encapsulation


    Materials and Methods

    Establishment of Stock Cultures, Multiplication and Shoot TipExcision. Ripe fruits were collected from a single sour orange tree innorth Jordan. Seeds were extracted and soaked in water for 12 h. Seeds weresurface-sterilized in 10% Clorox (5.25% sodium hypochlorite) solution for10 min, dipped in 70% alcohol with shaking for 30 s, and finally rinsed for5 min with distilled water three times. Nucellar embryos of seeds were thengerminated on a medium containing 0.1 M sucrose and 1.4mM gibberellicacid (GA3) and solidified with 7.5 g l

    21 Difco Bacto agar. Cultures weremaintained in the dark until germination, after which they were transferredto a growth room and maintained at 22 ^ 18C and 16 h light (photosyntheticphoton flux density, PPFD 5060mmol m22 s21=8 h dark. The shoots ofthe seedlings were cultured on solid MS (Murashige and Skoog, 1962)medium containing 0.1 M sucrose, 2.2mM 6-benzylaminopurine (BA), and0.6mM indole-3-acetic acid (IAA) and maintained under the growth roomconditions. Microshoots formed were subcultured every 4 wk on solid MSproliferation medium containing 0.1 M sucrose, 4.4mM BA, and 0.6mM IAAuntil enough mother stock culture was available.

    Shoot tips at the same developmental stage with expanding leaves wereexcised from the microshoots and placed in Petri dishes. With the aid of fine-end forceps and a needle, shoot tips were dissected under a binocularmicroscope to the size of 13 mm with two or three non-expanded leafprimordia.

    EncapsulationDehydration. Excised shoot tips were precultured onsolid MS medium containing 0.3 M sucrose for 1 wk. Shoot tips were thensuspended in calcium-free liquid MS medium supplemented with 3% (w/v)Na-alginate (2% viscosity kelp Na+ salt) solution and 0.1 M sucrose. Using a1 ml syringe, individual shoot tips were captured with some alginate solutionand dispensed as drops into standard liquid MS medium containing 100 mMcalcium chloride (CaCl2) and 0.1 M sucrose. After encapsulation, excessCaCl2 solution was poured off and the beads were left to polymerize for30 min. The encapsulated shoot tips were transferred to dehydrationsolutions containing liquid MS medium supplemented with 0.3, 0.5, or

    0.75 M sucrose for 2 d