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

CRYOPRESERVATION OF SOUR ORANGE (CITRUS AURANTIUM L.) SHOOT TIPS

SAMIA S. AL-ABABNEH, NABILA S. KARAM, AND RIDA A. SHIBLI*

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)

Summary

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

Cryopreservation was carried out via encapsulation–dehydration, vitrification, and encapsulation–vitrification 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 encapsulation–vitrification, 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.

Introduction

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 encapsulation–dehydration, vitrification, or

encapsulation–vitrification.

With encapsulation–dehydration, 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 reduces

the 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 10–30 min at 258C (Ashmore,

1997).

Encapsulation–vitrification, 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.

edu.jo

In Vitro Cell. Dev. Biol.—Plant 38:602–607, November–December 2002 DOI: 10.1079/IVP2002349q 2002 Society for In Vitro Biology1054-5476/02 $10.00+0.00

602

containing 2 M glycerol and 0.4 M sucrose during the encapsulation

process and then hydrated with PVS2 for 2–3 h (Matsumoto et al.,

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

encapsulated shoot tips to the vitrification solution at 08C is needed

to 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 encapsulation–dehydration

(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

encapsulation–dehydration, vitrification, and encapsulation–

vitrification.

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 l21 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 ¼ 50–60mmol m22 s21Þ=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 1–3 mm with two or three non-expanded leafprimordia.

Encapsulation–Dehydration. 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. For dehydration, encapsulated shoot tips were placedin uncovered Petri dishes at 23 ^ 18C and ambient RH (55–65%) 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).

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.

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.

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.

Encapsulation–Vitrification. 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. Encapsulated–vitrified 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.

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.

CRYOPRESERVATION OF SOUR ORANGE SHOOT TIPS 603

Results and Discussion

Encapsulation–Dehydration. There was a significant ðP #

0:01Þ interaction effect of sucrose concentration and dehydration

duration on survival and regrowth of cryopreserved shoot tips (Table

1). The greatest survival (83%) and regrowth (47%) were exhibited

by shoot tips that were precultured with 0.5 M sucrose and

dehydrated for 6 h, by which time the moisture content of beads was

18% (Table 1). Encapsulated cryopreserved shoot tips did not

survive without air dehydration. This may be attributed to formation

of extra- and intracellular ice crystals as a result of high moisture

content (Plessis et al., 1993) which was estimated to be 75–86% in

non-dehydrated tissue. Reduced moisture content is reported to be

essential for cryopreservation (Shibli et al., 2001). Differential

scanning calorimetry of encapsulated hop (Humulus lupulus ) shoot

tips dehydrated to different moisture contents showed a positive

correlation between desiccation and shoot tip survival after

cryopreservation (Martinez and Revilla, 1998). Maximum survival

of Holostemma annulare was achieved at 15.8–18.5% moisture

content (Decruse et al., 1999), whereas 28–33% moisture content

resulted in maximum recovery of olive (Olea europaea L.) shoot tips

(Martinez et al., 1999). In the current study, survival and regrowth

of encapsulated cryopreserved shoot tips increased with increasing

sucrose concentration to 0.75 M after 4 h of dehydration (Table 1).

Increased sucrose concentration in the pregrowth medium leads to

accumulation of solute inside the cells, resulting in maintaining

integrity of plasma and inner membranes during dehydration and

freezing (Plessis et al., 1993) and avoidance of ice crystals during

cooling and thawing (Grospietsch et al., 1999). Increased sucrose

concentration also leads to reduced moisture content of the

encapsulated shoot tips (Shibli et al., 1999; Tahtamouni and Shibli,

1999). Cell tolerance of dehydration and subsequent cooling in

liquid nitrogen was achieved after several days of preculture with a

high concentration of sucrose (Bachiri et al., 1995). After 6 h of

dehydration in the current study, survival and regrowth increased

with increasing sucrose concentration to 0.5 M but declined with

0.75 M (Table 1), which may be attributed to osmotic stress. The

reduction in regrowth compared with survival may be attributed to

partial damage of the shoot tips due to osmotic shock after

rehydration and ice crystallization of some cells in the shoot tips.

A significant interaction effect of duration of dehydration with

silica gel and cyropreservation with liquid nitrogen on survival

ðP # 0:05Þ and regrowth ðP # 0:01Þ of shoot tips was detected

(Table 2). Maximum survival (100%) and regrowth (80%) was

achieved when encapsulated non-cryopreserved shoot tips were

dehydrated with silica gel for 2 h. Although the shoot tips were

green with 4 h dehydration, there was reduction in survival (73%)

and regrowth (60%) due to damage occurring during extended

dehydration. When encapsulated cryopreserved shoot tips were

exposed to 2 h dehydration with silica gel, 93% of the shoot tips

survived but only 37% regrew and the tips were yellow (Table 2).

With greater dehydration, survival of encapsulated cryopreserved

shoot tips was either complete with low (10%) regrowth or low

(60%) with complete loss of regrowth. Tips of recovered shoots also

developed necrosis. The inability of cryopreserved shoot tips to

regrow after dehydration with silica gel for 4 h and then exposure

to liquid nitrogen may be due to over-dehydration, which leads to

inability of shoot tips to rehydrate without cellular damage after

thawing.

Vitrification effect of sucrose concentration and cyroprotectant on

survival of non-cryopreserved and cryopreserved shoot tips. There

was a significant ðP # 0:01Þ interaction effect of sucrose

concentration in the preculture medium, cryoprotectant, and

cryopreservation with liquid nitrogen on survival of shoot tips

(Table 3). The greatest survival percentage (97%) was obtained

when non-cryopreserved shoot tips were precultured with 0.5 M

sucrose compared to a maximum of 67% survival for shoots

precultured with 0.75 M sucrose. Non-cryopreserved shoot tips

exhibited high survival frequencies (87–97%) after preculture with

0.5 M sucrose irrespective of the cryoprotectant type. However, with

the same preculture medium, cryopreserved shoot tips exhibited

variations in survival depending on the cryoprotectant used; 2 M

sucrose plus 10% DMSO being the most effective (80% survival).

When the preculture medium was supplemented with 0.75 M

sucrose, all cryopreserved shoot tips exhibited low survival

frequencies (10–37%), whereas non-cryopreserved ones exhibited

maximum (67%) survival when 1 M sucrose was used as a

TABLE 1

SURVIVAL AND REGROWTH OF ENCAPSULATED–DEHYDRATEDAND CRYOPRESERVED CITRUS AURANTIUM SHOOT TIPS

(PRECULTURED ON SOLID MS MEDIUM CONTAINING 0.3 MSUCROSE FOR 1 WK) AS INFLUENCED BY 2-d DEHYDRATION IN

LIQUID MS MEDIUM CONTAINING DIFFERENT CONCENTRATIONSOF SUCROSE FOLLOWED BY AIR DEHYDRATION FOR DIFFERENT

DURATIONS

Sucroseconcentration(M )

Dehydration(h)

Moisturecontent

(%) Survival (%) Regrowth (%)

0.3 0 85.8 0 f 0 f4 21.8 23 e 13 e6 21.8 30 e 20 de

0.5 0 82.4 0 f 0 f4 22.5 56 c 30 bc6 18.0 83 a 47 a

0.75 0 74.9 0 f 0 f4 19.2 70 b 37 b6 17.5 47 d 23 cd

Means within columns having different letters are significantly differentaccording to LSD ðP # 0:01Þ:

TABLE 2

INFLUENCE OF DURATION OF DEHYDRATION WITH SILICA GEL ONSURVIVAL AND REGROWTH OF NON-CRYOPRESERVED (2LN) AND

CRYOPRESERVED (þLN) CITRUS AURANTIUM SHOOT TIPSPRECULTURED ON SOLID MS MEDIUM CONTAINING 0.3 M SUCROSE

FOR 1 WK

Dehydration duration (h) Survival (%) Regrowth (%)

2 2LN 100 a 80 aþLN 93 a 37 d

3 2LN 100 a 50 cþLN 100 a 10 e

4 2LN 73 b 60 bþLN 60 c 0 f


604 AL-ABABNEH ET AL.

cryoprotectant (Table 3). Differences observed in survival after

exposure to different cryoprotectant combinations may be due to

differences in permeability of the cryoprotectant inside the plant

tissue, ability to induce osmotic stress, and toxic effects. Increased

concentration of the cryoprotectant in the medium leads to reduced

survival percentage due to its toxic effect at higher concentrations

(Reed, 1995). Rajasekaran (1996) reported that cells of cotton

(Gossypium hirsutum L.) frozen with DMSO alone took longer to

regrow, which may be due to the fact that DMSO alone is toxic to

cells. Gazeau et al. (1998) found that using DMSO as a

cryoprotectant was effective in increasing intracellular viscosity

and thus avoiding formation of ice crystals.

Effect of loading and vitrification solutions on survival and

regrowth of non-cryopreserved and cryopreserved shoot tips. A

significant ðP # 0:01Þ interaction effect of the loading solution,

cryoprotectant, and cryopreservation with liquid nitrogen on

survival and regrowth of shoots tips was detected (Table 4). The

greatest survival (100%) and regrowth (73%) were obtained when

non-cryopreserved shoot tips were loaded with 1 M sucrose plus 5%

DMSO and cryoprotected with 1 M sucrose plus 30% DMSO. With

cryopreserved shoot tips, using 0.4 M sucrose plus 2 M glycerol as a

loading solution and 100% PVS2 as a cryoprotectant resulted in

maximum survival of 67% and only 43% regrowth. With the loading

solution 1 M sucrose plus 5% DMSO, survival of cryopreserved

shoot tips was low (17–27%) and regrowth was completely lost

irrespective of the cryoprotectant used (Table 4). The loading phase

is necessary to reduce osmotic shock caused by direct exposure of

precultured shoot tips to concentrated 100% PVS2 (Sarker and

Naik, 1998).

Survival and regrowth of frozen shoot tips were less than those of

unfrozen ones which may be attributed to formation of intracellular

ice crystals during freezing and/or thawing (Matsumoto et al., 1994)

as a result of insufficient dehydration of shoot tips (Sakai et al.,

2000). Sakai et al. (1991) demonstrated that complete vitrification

of the cryopreserved plant tissues eliminates concern for the

potentially damaging effects of intra- and extracellular crystal-

lization. Increased concentration of the vitrification solution up to

an optimal level and increased duration of exposure to the

vitrification solution led to increased solute concentration inside the

plant tissues (Bachiri et al., 1995). Regardless of cryopreservation

or type of loading or cryoprotectant solution used in the current

study, regrowth percentages were lower than survival percentages

(Table 4), indicating that not all surviving shoot tips were able to

regrow. This may be due to the fact that only a localized group of

cells in the leaf primordium tissue or the meristematic dome area

remained alive after the stress of freezing and thawing (Gonzalez-

Arnao et al., 1993).

Effect of duration of exposure to the loading solution on survival

and regrowth of non-cryopreserved and cryopreserved shoot

tips. There was a significant ðP # 0:01Þ interaction effect of

duration of exposure to the loading solution (0.4 M sucrose plus 2 M

glycerol) and cryopreservation with liquid nitrogen on survival and

regrowth of shoot tips (Table 5). Exposure of non-cryopreserved

shoot tips to the loading solution for 60 min resulted in 97%

survival and 100% regrowth of shoots. On the other hand, a

maximum of 83% survival and only 47% regrowth of cryopreserved

shoot tips was obtained after exposure to the loading solution for

60 min. This may be due to formation of intracellular ice crystals

after liquid nitrogen exposure (Gonzalez-Arnao et al., 1998). Direct

exposure of sucrose precultured shoot tips to concentrated PVS2 is

detrimental to the viability of vitrified shoot tips, and sufficient time

should be available to enhance solute permeation into the

cytoplasm (Sarker and Naik, 1998). Sakai et al. (1990) reported

that 20 min exposure to loading solutions was sufficient to reduce

TABLE 3

SURVIVAL (%) OF NON-CRYOPRESERVED (2LN) ANDCRYOPRESERVED (þLN) CITRUS AURANTIUM SHOOT TIPS AS

INFLUENCED BY SUCROSE CONCENTRATION IN THE PRECULTUREMEDIUM (1 d) AND CRYOPROTECTANT TYPE

Sucrose concentration in preculturemedium (M )

0.5 0.75

Cryoprotectant 2LN þ LN 2LN þ LN

1 M sucrose 87 ab 23 ghi 67 c 20 hij2 M sucrose 90 ab 13 ij 47 d 13 ij1 M sucrose þ 5% DMSO 97 a 13 ij 43 de 37 def1 M sucrose þ 10% DMSO 87 ab 40 def 33 efg 13 ij2 M sucrose þ 5% DMSO 90 ab 43 de 40 def 30 fgh2 M sucrose þ 10% DMSO 90 ab 80 b 20 hij 10 j

Means having different letters are significantly different according to LSDðP # 0:01Þ:

TABLE 4

INFLUENCE OF TYPE OF LOADING AND CRYOPROTECTANT SOLUTIONS ON SURVIVAL AND REGROWTH OF NON-CRYOPRESERVED (2LN)AND CRYOPRESERVED (þLN) CITRUS AURANTIUM SHOOT TIPS PRECULTURED ON SOLID MS MEDIUM SUPPLEMENTED WITH 0.5 M SUCROSE

FOR 1 d

Loading solution Cryoprotectant Survival (%) Regrowth (%)

0.4 M sucrose þ 2 M glycerol 100% PVS2 2LN 77 b 70 aþLN 67 bc 43 c

0.4 M sucrose þ 2 M glycerol 1 M sucrose þ 30% DMSO 2LN 90 a 50 cþLN 60 c 30 d

1 M sucrose þ 5% DMSO 100% PVS2 2LN 77 b 60 bþLN 27 d 0 e

1 M sucrose þ 5% DMSO 1 M sucrose þ 30% DMSO 2LN 100 a 73 aþLN 17 d 0 e

Means within columns having different letters are significantly different according to LSD ðP # 0:01Þ:


the toxic effect of concentrated PVS2. In the current study,

exposure to the loading solution for longer than 60 min resulted in

significant reduction in survival and regrowth (Table 5), which may

be attributed to increased osmotic stresses and chemical toxicity of

the vitrification solution (Sakai et al., 1990).

Encapsulation–Vitrification. Survival and regrowth of encapsu-

lated–vitrified and cryopreserved shoot tips were only 50–57% and

30–40%, respectively (Table 6). Tips of the recovered shoots were

yellow or pale green irrespective of duration of exposure to PVS2,

which may be attributed to cellular changes of some tissues in the

cryopreserved shoot tips (Paul et al., 2000). Encapsulation of shoot

tips before exposure to a vitrification solution at 08C is needed to

reduce the injurious effect of the vitrification solution and reduce

cell permeability, thus increasing the length of time that cells are

exposed to the osmotic stress (Sakai et al., 1991). By using

encapsulation–vitrification, tolerance to dehydration and freezing is

achieved through capsule protection and osmoprotection with a

vitrification solution (Hirai and Sakai, 1999). In the current study,

maximum survival (80%) and regrowth (77%) of encapsulated–

vitrified non-cryopreserved shoot tips were obtained after 2 h

dehydration with concentrated PVS2 at 08C (Table 6). Hirai and

Sakai (1999) reported that the greatest shoot formation was obtained

after dehydrating encapsulated shoot tips of mint (Mentha spicata

L.) with 100% PVS2 for 3 h at 08C. However, Matsumoto et al.

(1995) found that maximum shoot formation of encapsulated wasabi

(Wasabia japonica ) was obtained after 70–100 min dehydration

at 08C.

Conclusion

Reliable protocols for cryopreservation of sour orange (Citrus

aurantium ) shoot tips were developed for the first time using

encapsulation–dehydration, vitrification and encapsulation–

vitrification procedures. These protocols are vital for base gene

banking of sour orange to ensure future availability of this valuable

rootstock. Although encapsulation–dehydration outperformed other

treatments and achieved maximum (93%) survival, maximum (43%)

regrowth was obtained with vitrification, whereas encapsulation–

vitrification was intermediate.

Acknowledgments

The authors would like to thank the Deanship of Research at JordanUniversity of Science and Technology for funding this study, Project #(237/99). The technical assistance of Mr. Mohammad Shatnawi is greatlyappreciated.

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TABLE 5

INFLUENCE OF DURATION OF EXPOSURE TO THE LOADINGSOLUTION (0.4 M SUCROSE PLUS 2 M GLYCEROL) ON SURVIVAL

AND REGROWTH OF NON-CRYOPRESERVED (2LN) ANDCRYOPRESERVED (þLN) CITRUS AURANTIUM SHOOT TIPS

PRECULTURED ON SOLID MS MEDIUM SUPPLEMENTED WITH 0.5 MSUCROSE FOR 1 d

Durationof exposure(min) Survival (%) Regrowth (%)

25 2LN 93 a 93 aþLN 60 d 30 d

30 2LN 80 b 73 bþLN 60 d 50 c

60 2LN 97 a 100 aþLN 83 b 47 c

90 2LN 70 c 47 cþLN 47 e 20 d


TABLE 6

INFLUENCE OF DURATION OF DEHYDRATION WITH 100% PVS2 ONSURVIVAL AND REGROWTH OF NON-CRYOPRESERVED (2LN) AND

CRYOPRESERVED (þLN) CITRUS AURANTIUM SHOOT TIPSPRECULTURED ON SOLID MS MEDIUM CONTAINING 0.3 M SUCROSE

FOR 1 d

Dehydration duration (h) Survival (%) Regrowth (%)

2 2LN 80 a 77 aþLN 50 b 30 c

3 2LN 70 a 60 bþLN 57 b 40 c


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