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

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<ul><li><p>CRYOPRESERVATION OF SOUR ORANGE (CITRUS AURANTIUM L.) SHOOT TIPS</p><p>SAMIA S. AL-ABABNEH, NABILA S. KARAM, AND RIDA A. SHIBLI*</p><p>Department of Plant Production, Faculty of Agriculture, Jordan University of Science and Technology, P.O. Box 3030, Irbid, Jordan</p><p>(Received 23 November 2002; accepted 5 July 2002; editor S. A. Merkle)</p><p>Summary</p><p>The objective of this study was to establish a cryopreservation protocol for sour orange (Citrus aurantium L.).</p><p>Cryopreservation was carried out via encapsulationdehydration, vitrification, and encapsulationvitrification on shoot</p><p>tips excised from in vitro cultures. Results indicated that a maximum of 83% survival and 47% regrowth of encapsulated</p><p>dehydrated and cryopreserved shoot tips was obtained with 0.5 M sucrose in the preculture medium and further</p><p>dehydration for 6 h to attain 18% moisture content. Dehydration of encapsulated shoot tips with silica gel for 2 h resulted</p><p>in 93% survival but only 37% regrowth of cryopreserved shoot tips. After preculturing with 0.5 M sucrose, 80% of the</p><p>vitrified cryopreserved shoots survived when 2 M sucrose plus 10% dimethyl sulfoxide (DMSO) was used as a</p><p>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</p><p>vitrification solution (PVS2) for 20 min. Increased duration of exposure to the loading solution up to 60 min increased</p><p>survival (83%) and regrowth (47%) of cryopreserved shoot tips. With encapsulationvitrification, dehydration with 100%</p><p>PVS2 for 2 or 3 h at 08C resulted in 50 or 57% survival and 30 or 40% regrowth, respectively, of cryopreserved shoot tips.</p><p>Key words: preservation; cryopreservation; encapsulation; vitrification.</p><p>Introduction</p><p>Cryopreservation in liquid nitrogen (21968C) is a preservation</p><p>method in which cell division and metabolic and biochemical</p><p>processes are arrested (Niino and Sakai, 1992). Thus, the plant</p><p>material is stored without deterioration or modification for an</p><p>unlimited time (Lambardi et al., 2000) and genetic stability and</p><p>regeneration potential of the cryopreserved material are</p><p>maintained (Rajasekaran, 1996). Cryopreservation may be</p><p>achieved through encapsulationdehydration, vitrification, or</p><p>encapsulationvitrification.</p><p>With encapsulationdehydration, explants are encapsulated in</p><p>beads, dehydrated, and then cooled rapidly in liquid nitrogen</p><p>(Bachiri et al., 1995; Niino et al., 1995; Shibli et al., 1999; Sakai</p><p>et al., 2000). This method is simple, inexpensive and the high</p><p>genetic stability of the cryopreserved material can be maintained</p><p>(Kartha and Engelmann, 1994).</p><p>In vitrification, tissues are dehydrated with a highly concentrated</p><p>osmoticum to avoid ice formation during cryopreservation and</p><p>thawing (Bachiri et al., 1995). This technique is simple, does not</p><p>need expensive cooling apparatus, and can be applied to a wide</p><p>range of plant material (Niino and Sakai, 1992; Matsumoto et al.,</p><p>1994). The technique consists of three major phases (Engelmann,</p><p>1997; Tahtamouni and Shibli, 1999). The loading phase involves</p><p>treatment of tissue with cryoprotectants or diluted vitrification</p><p>solutions (Ashmore, 1997). The dehydration phase involves</p><p>dehydrating plant tissue with a highly concentrated vitrification</p><p>solution (Sakai et al., 1991). The plant vitrification solution is an</p><p>aqueous cryoprotectant solution in which living systems can be</p><p>cooled slowly without appreciable intra- or extracellular ice</p><p>formation (Fahy et al., 1987). This solution increases the osmotic</p><p>potential of the external medium (Reed, 1995), resulting in flow of</p><p>water out of the cells and dehydration of tissue (Ashmore, 1997). A</p><p>single cryoprotectant, usually dimethyl sulfoxide (DMSO), is</p><p>effective (Goldner et al., 1991) although a cryoprotectant mixture</p><p>may be more effective for some plant species (Sakai et al., 1990).</p><p>High concentrations of cryoprotectants in the medium lead to</p><p>reduced survival due to their toxic effect (Reed, 1995). The duration</p><p>of contact between the explant and the vitrification solution is a</p><p>critical parameter affecting the survival percentage of the</p><p>cryopreserved plant material (Engelmann, 1997). The dehydration</p><p>period generally increases with the size of the explant used</p><p>(Ashmore, 1997). Permeating the dehydration step at 08C reducesthe vitrification solution toxicity (Ashmore, 1997), thus broadening</p><p>duration of exposure to the vitrification solution and increasing</p><p>survival percentage of the cryopreserved plant tissues (Engelmann,</p><p>1997). The unloading phase starts after rapid warming, where plant</p><p>vitrification solution (PVS2) is drained out of the cryotubes and</p><p>replaced with 1.2 M sucrose for 1030 min at 258C (Ashmore,</p><p>1997).</p><p>Encapsulationvitrification, which is a combination of encapsu-</p><p>lation and vitrification (Engelmann, 1997), reduces the injury effect</p><p>of the vitrification solution on explants (Ashmore, 1997) and results</p><p>in higher survival rates (Hirai and Sakai, 1999). With this</p><p>technique, the plant material is osmoprotected with a mixture*Author to whom correspondence should be addressed: Email shibli@just.</p><p></p><p>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</p><p>602</p></li><li><p>containing 2 M glycerol and 0.4 M sucrose during the encapsulation</p><p>process and then hydrated with PVS2 for 23 h (Matsumoto et al.,</p><p>1995; Hirai and Sakai, 1999; Sakai et al., 2000). Exposing</p><p>encapsulated shoot tips to the vitrification solution at 08C is neededto reduce the injurious effect of the vitrification solution and thus</p><p>the time that cells are exposed to osmotic stress may be extended</p><p>(Hirai and Sakai, 1999). With this technique, dehydration and</p><p>freezing tolerance is achieved through capsules osmoprotected with</p><p>vitrification solutions (Hirai and Sakai, 1999; Shibli and</p><p>Al-Juboory, 2000). This technique is easy to handle, saves the</p><p>time needed for dehydration (Hirai et al., 1998; Sakai et al., 2000;</p><p>Shibli and Al-Juboory, 2000), and results in growth recovery which</p><p>is much earlier than that with encapsulationdehydration</p><p>(Matsumoto et al., 1995; Hirai and Sakai, 1999). Encapsulation</p><p>vitrification has been described as a cryogenic protocol with high</p><p>potential for large-scale cryopreservation (Hirai et al., 1998; Shibli</p><p>and Al-Juboory, 2000).</p><p>Sour orange (Citrus aurantium L.) is used as a rootstock and has</p><p>several advantages over the commercially used seedling rootstocks,</p><p>including resistance to several viral diseases and improvement of</p><p>fruit quality of the grafted species (Samson, 1986). Sour orange is</p><p>endangered and the use of other rootstocks will result in a decline in</p><p>performance of sour orange rootstock with time due to unfavorable</p><p>environmental conditions, especially drought and salinity. This</p><p>mandates preservation of valuable genetic resources of sour orange</p><p>for future use and improvement. Therefore, this study was initiated</p><p>to develop protocols for cryopreservation of sour orange via</p><p>encapsulationdehydration, vitrification, and encapsulation</p><p>vitrification.</p><p>Materials and Methods</p><p>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</p><p>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.</p><p>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.</p><p>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</p><p>0.75 M sucrose for 2 d. For dehydration, encapsulated shoot tips were placedin uncovered Petri dishes at 23 ^ 18C and ambient RH (5565%) and heldfor 0, 4, or 6 h within the laminar air flow cabinet. Some encapsulated shoottips were placed on sterile filter paper and dehydrated in Parafilm-sealedPetri dishes containing 17 g of sterile silica gel for 2, 3, or 4 h. Dehydratedencapsulated shoot tips were then transferred to sterile cryovials andplunged directly into liquid nitrogen for a minimum of 30 min. The cryovialswere removed from liquid nitrogen and warmed in a water bath at 358C for2 min. The beads were then cultured on MS solid medium supplemented with0.9mM BA and maintained in the dark in the growth room for 3 d. Survivalwas evaluated for part of the examined cryopreserved shoot tips bytetrazolium chloride (TTC) test (Shibli et al., 2001). The remaining shoot tipswere transferred to the growth room. Two weeks later, the encapsulated shoottips were examined under a binocular microscope for any sign of growth.Empty beads were processed as described before to determine the change inmoisture content with dehydration time (Shibli et al., 2001).</p><p>Vitrification effect of sucrose concentration and cyroprotectant on survivalof non-cryopreserved and cryopreserved shoot tips. Excised shoot tips wereprecultured on solid MS medium supplemented with 0.5 or 0.75 M sucrosefor 1 d under the growth conditions described previously. The shoot tips wereplaced in cryovials containing 1 ml of a cryoprotectant solution for 20 min at258C. The cryoprotectant solution was liquid MS medium supplemented with(1) 1 M sucrose, (2) 2 M sucrose, (3) 1 M sucrose and 5% DMSO, (4) 1 Msucrose and 10% DMSO, (5) 2 M sucrose and 5% DMSO, or (6) 2 M sucroseand 10% DMSO. A number of shoot tips were then placed in cryovials whichwere plunged into liquid nitrogen for 30 min and thawed for 3 min in awater bath at 388C. The cryoprotectant solution was removed from non-cryopreserved and cryopreserved shoot tips and replaced with unloadingsolution (1.2 M sucrose) for 10 min. After removing the unloading solution,treated shoot tips were subcultured on recovery medium supplemented with0.3 M sucrose and incubated in the dark for 1 d. Data were collected onsurvival after 2 d of transferring shoot tips to MS media using TTC test.Recovery and leaf color were monitored over 3 wk.</p><p>Effect of loading and vitrification solutions on survival and regrowth ofnon-cryopreserved and cryopreserved shoot tips. Excised shoot tips wereprecultured on solid MS medium supplemented with 0.5 M sucrose for 1 d.Shoot tips were then placed in cryovials containing 1 ml of one of two loadingsolutions (liquid MS medium supplemented with 0.4 M sucrose and 2 Mglycerol, or 1 M sucrose and 5% DMSO) for 20 min at 258C. The loadingsolution was replaced with one of two vitrification solutions (liquid MSmedium supplemented with 100% PVS2 solution, or 1 M sucrose plus 30%DMSO) for 20 min at 258C. Thawing and unloading were performed asdescribed previously. After washing with the unloading solution, shoottips were subcultured on recovery medium supplemented with 0.3 Msucrose and incubated in the dark for 1 d. Survival was examined for part ofthe shoot tips. Other shoot tips were recultured on fresh recovery mediumcontaining 0.9mM BA and incubated in the dark for 3 d after which theywere transferred to the growth room. Data were collected as mentionedearlier.</p><p>Effect of duration of exposure to the loading solution on survival andregrowth of non-cryopreserved and cryopreserved shoot tips. Excised shoottips were precultured on solid MS medium supplemented with 0.5 M sucrosefor 1 d. The precultured shoot tips were placed in cryovials and loaded with1 ml of a loading solution (0.4 M sucrose and 2 M glycerol) for 25, 30, 60, or90 min at 258C. The loading solution was then replaced with 1 ml of 100%PVS2 for 10 min at 258C. This solution was replaced with PVS2 for 10 min at08C. Shoot tips were tested for survival and regrowth before and after dippingin liquid nitrogen as described previously.</p><p>EncapsulationVitrification. Excised shoot tips were precultured onsolid MS medium containing 0.3 M sucrose for 1 d. Precultured shoot tipswere encapsulated in alginate beads supplemented with 0.4 M sucrose and2 M glycerol. Encapsulated shoot tips were vitrified with 100% PVS2 for 2 or3 h at 08C. Encapsulatedvitrified shoot tips were placed in cryovialsand plunged rapidly in liquid nitrogen for 30 min. Thawing, unloading,incubation and reculturing were performed as described earlier. Data werecollected as described previously.</p><p>Experimental Design and Statistical Analysis. Treatments in the above-described experiments were arranged in a completely randomized design.Each treatment was replicated three times with 10 shoot tips per replicate.Data were analyzed as a repeated measure using MSTATC software(Michigan State University, 1988). Means were separated according to theleast significant difference (LSD) method at 0.01 level of probability.</p><p>CRYOPRESERVATION OF SOUR ORANGE SHOOT TIPS 603</p></li><li><p>Results and Discussion</p><p>EncapsulationDehydration. There was a significant P #0:01 interaction effect of sucrose concentration and dehydrationduration on survival and regrowth of cryopreserved shoot tips (Table</p><p>1). The greatest survival (83%) and regrowth (47%) were exhibited</p><p>by shoot tips that were precultured with 0.5 M sucrose and</p><p>dehydrated for 6 h, by which time the moisture content of beads was</p><p>18% (Table 1). Encapsulated cryopreserved shoot tips did not</p><p>survive without air dehydration. This may be attributed to formation</p><p>of extra- and intracellular ice crystals as a result of high moisture</p><p>c...</p></li></ul>


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