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Journal of Plant Physiology 166 (2009) 1923—1933

Physiological and growth changes inmicropropagated Citrus macrophyllaexplants due to salinity

Olaya Perez-Tornero�, Carlos I. Tallon, Ignacio Porras, Josefa M. Navarro

Departamento de Citricultura, Instituto Murciano de Investigacion y Desarrollo Agrario y Alimentario, C/Mayor s/n.30150 La Alberca, Murcia, Spain

Received 25 February 2009; received in revised form 3 June 2009; accepted 5 June 2009

KEYWORDSIn vitro culture;Malondialdehyde;Mineral nutrition;Proline;Quaternaryammoniumcompounds

SummarySalinity is one of the major abiotic stresses affecting arable crops worldwide, and isthe most stringent factor limiting plant distribution and productivity. In the presentstudy, the possible use of in vitro culture to evaluate the growth and physiologicalresponses to salt-induced stress in cultivated explants of Citrus macrophylla wasanalyzed. For this purpose, micropropagated adult explants were grown inproliferation and rooting media supplemented with different concentrations ofNaCl. All growth parameters were decreased significantly by these NaCl treatments;this was accompanied by visible symptoms of salt injury in the proliferated shootsfrom 60mM NaCl and in the rooted shoots from 40mM NaCl. Malondialdehyde (MDA)increased with increasing salinity in proliferated shoots, indicating a rising degree ofmembrane damage. The concentration of total chlorophyll significantly decreased inthe presence of NaCl, and this effect was more pronounced in the rooted explants.The Na+ and Cl� concentrations in the explants increased significantly with thesalinity level, but Cl� levels were higher in the proliferated explants than in therooted explants. For osmotic adjustment, high concentrations of compatible solutes(proline and quaternary ammonium compounds—QAC) accumulated in salt-stressedplants in proliferation, but differences were not observed in rooted explants. Inproliferation, proline and QAC were highly correlated with the sodium and chlorideconcentrations in the explants, indicating a possible role of these compounds in

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Abbreviations: BA, 6-benzylaminopurine; GA, gibberellic acid; IBA, indole-butyric acid; IAA, indole-acetic acid; MDA,malondialdehyde; QAC, quaternary ammonium compounds.�Corresponding author. Tel.: +34 968366757; fax: +34 968366792.E-mail address: olalla.perez@carm.es (O. Perez-Tornero).

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osmotic adjustment. The plant concentrations of NO3�, K+, Mg2+, Ca+ and Fe were

also affected by the NaCl concentration of the medium. We suggest that theimportant deleterious effects in the in vitro explants of Citrus macrophylla grown atincreasing NaCl concentrations were due mainly to toxic effects of saline ions,particularly Cl�, at the cellular level.& 2009 Elsevier GmbH. All rights reserved.

Introduction

Salinity is one of the major abiotic stresses thathave adverse effects on the growth of plants andthe productivity of crops. These abiotic stresses arebecoming even more prevalent as the intensity ofagriculture increases (Zhu, 2002). Therefore, elu-cidation of the mechanisms of tolerance at specificstages of plant development is critical to under-standing the plant response and to achievinggenetic or environmental improvement of stresstolerance (Zhu, 2001). Citrus is produced in aridclimates, where salts occur naturally in soils andare being introduced in irrigation waters andfertilizers. Consequently, salinity damage is aproblem in citrus production (Ferguson andGrattan, 2005). Compared with other crops, citrusspecies are among the most sensitive to soilsalinity, although the ability of citrus trees totolerate salinity varies among species and dependson the rootstock (Maas, 1993). Therefore, studiesof the mechanism of salinity tolerance in citrus aremuch needed.

Evaluating field performance crops under salineconditions is notoriously difficult because of thevariability of salinity within fields (Daniells et al.,2001) and the enormous potential for interactionswith other environmental factors. In vitro tissueculture is a simple system that offers a suitablealternative to studying physiological mechanisms oftolerance to salinity, since it provides relativelyfast responses, a short generation time and acontrolled environment, especially in tree speciesthat have long reproductive cycles (Torregrosa andBouquet, 1993). A close relationship between the invitro and in vivo responses to salt has been found indifferent species: in grapevine rootstocks (Troncosoet al., 1999) and in mulberry genotypes withdiffering salt tolerance (Vijayan et al., 2003).

Tissue culture techniques using plant tissues orcells are good tools for salinity studies, and thepotential for physiological, nutritional and growthresponses to differentiate between salt-sensitiveand salt-tolerant species or individuals is beingexamined. Using irradiated embryogenic suspen-sion cultures, He et al. (2009) selected salt-tolerant lines of sweet potato with higher proline

levels and superoxide dismutase activity. Queiroset al. (2007) selected different NaCl-tolerant celllines of potato with higher lipid peroxidation,ascorbic acid and insoluble proteins. Although theresponse of woody species to salt exposure in tissueculture has received little attention, it is gaininginterest. Troncoso et al. (1999) found that theNaCl-tolerance of in vitro-grown grapevine root-stocks is due to their capacity to accumulate salt,to increase K+ levels and to maintain a high watercontent. Woodward and Bennett (2005) observedthat, in Eucalyptus camaldulensis cultivated invitro, proline is accumulated in salt-tolerantclones.

Citrus macrophylla is used widely in Spain as arootstock for lemons, because the otherwise-pre-ferred rootstock, ‘Sour Orange’, is sensitive to thetristeza virus (Moreno et al., 2008). In the presentinvestigation, we report the response of Citrusmacrophylla explants cultured in vitro to differentNaCl concentrations, in relation to several growth,nutritional and physiological parameters. Althoughdifferent studies have been carried out, both underfield and greenhouse conditions, there are very fewreports of the in vitro salt tolerance of citrus and,to the best of our knowledge, this is the first studyof the in vitro salt tolerance of Citrus macrophyllaadult explants. The objective of our work was tostudy the use of in vitro culture to evaluate theresponses to salt-induced stress in cultivatedexplants of Citrus macrophylla. Specifically, weexamined the nutritional, physiological and growthresponses to increasing external NaCl concentra-tion, to study the different mechanisms used byproliferated and rooted explants.

Materials and methods

Plant material and medium composition

Two experiments were conducted, one withexplants in proliferation and another with explantsin rooting. Explants in proliferation were obtainedfrom previous in vitro cultures of Citrus macro-phylla in our laboratory, and maintained in the

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growth room. The standard proliferation mediumconsisted of macronutrients, micronutrients andvitamins of the DKW medium (Driver and Kuniyuki,1984) supplemented with 8.9 mM 6-benzylamino-purine (BA), 4.3 mM gibberellic acid (GA), 0.49 mMindole-butyric acid (IBA), 25mg L�1 phloroglucinol,30 g L�1 sucrose and 6 g L�1 agar (Hispanlab).

For the rooting experiment, explants weretransferred from the proliferation medium to therooting medium, which consisted of macronutri-ents, micronutrients and vitamins of the DKWmedium supplemented with 4.9 mM IBA, 5.7 mMindole-acetic acid (IAA), 25mg L�1 phloroglucinol,30 g L�1 sucrose and 6 g L�1 agar (Hispanlab).

After addition of plant growth regulators andadjustment of the pH to 5.7 with 1N NaOH, 100mLof each medium were dispensed into 500-mL jarsand sterilized in an autoclave at 121 1C for 21min.Cultures were grown at 2571 1C with white light(5000 lx) and a 16-h photoperiod.

Salinity treatments

For the proliferation treatments, salinity stresswas achieved by using shoot tips (1–1.5 cm long),from the proliferation medium, which were placedin the standard proliferation medium with sixdifferent concentrations of NaCl (0, 30, 60, 90,120 and 150mM). Each treatment consisted of fivereplicates (jars) with 12 shoots per jar. Theexplants were transferred to fresh medium every4 weeks.

For the rooting treatments, 20-mm shoot tips,from the proliferation medium, were used; thesewere placed in the standard rooting medium withsix different concentrations of NaCl (0, 20, 40, 60,80 and 100mM). The NaCl concentrations werelower than in the proliferation experiment be-cause, in previous experiments, no rooting wasobserved at higher concentrations. Each treatmentconsisted of four replicates (jars) with 10 shootsper jar.

Determination of growth parameters

Eight weeks after the beginning of the prolifera-tion experiment, the following growth parameterswere evaluated: number of shoots longer than5mm per explant, their length and productivity(number of shoots� the average shoot length) andthe fresh and dry weights.

For the rooting treatments, 4 weeks after thebeginning of the experiment, the rooting para-meters were evaluated: rooting percentage, shootlength, number of roots and their length, damaged

and fallen leaves and fresh and dry weights ofshoots and roots.

Determination of tissue mineralconcentrations

Leaves and stems were used for analyticaldeterminations. Plants were weighed fresh andafter freeze-drying. Dried material was thenground and stored for chemical analysis.

Dried and ground plant tissues were ashed anddissolved in HNO3 for determination of Na+, K+,Mg2+, Ca2+, P and Fe using an inductively coupledplasma optical emission spectrometer (Varian MPXVista). Chloride and NO3

� were extracted with bi-distilled water using the method of Guilliam (1971),and determined by ionic chromatography with aDionex ICS-3000 ion chromatograph equipped witha conductivity detector and an AS11-HC anionexchange column.

Determination of physiological parameters

Chlorophyll contents were estimated followingthe procedure described by Inskeep and Bloom(1985), by extracting 20mg of the ground materialwith N,N-dimethylformamide and measuring theabsorbance at 664.5 and 647 nm.

Lipid peroxidation was determined by measuringmalondialdehyde (MDA) using the Heath and Packer(1968) method. Dry tissue was homogenized with athiobarbituric acid–trichloracetic acid mixture.After 45min at 95 1C and centrifugation at 4000gfor 35min, the absorbance was read at 532 and600 nm for correction of nonspecific turbidity.

The quaternary ammonium compounds (QAC)were measured by the Grieve and Grattan (1983)method. Samples were extracted with deionizedH2O and, after dilution with 2N H2SO4, periodidecrystals were formed with cold KI-I2 reagent.Absorbance at 365 nm was measured after dissolu-tion of periodide crystals in 1,2-dichloroethane.

Free proline was analyzed by a modification ofthe acid-ninhydrin method (Bates et al., 1973).Samples were extracted with 5-sulphosalycilic acidat –15 1C for 12 h. After glacial acetic acid and acid-ninhydrin addition, the color was developed at100 1C and absorbance measured at 520 nm.

Statistical analysis

The data obtained from the different experi-ments were analyzed using the General LinearModel procedure in SPSS software (SPSS 13.0for Windows), employing ANOVA models. The

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significance of the differences between meanvalues was determined with the least significantdifference (LSD) test at the 5% level of probability.The values presented are means and their standarderrors. Linear correlations between the concentra-tions of Na+ and Cl� and those of proline and QAC inproliferation were calculated using the SPSS soft-ware package.

Results and discussion

Influence of NaCl on the growth of explants

After 8 weeks in the proliferation culture media,the growth parameters (shoot number, length,productivity and fresh and dry weights) weresignificantly affected (Po0.001, for all parameters)by the NaCl treatments (Table 1). Shiyab et al.(2003) also observed a significant decrease in thegrowth parameters at and above 150mM NaClin explants from seeds of sour orange (Citrus

aurantium) in proliferation. In explants of Citrusmacrophylla in proliferation, a significant decreasein the growth parameters was observed from 60mMNaCl, except for the shoot number, which de-creased from 30mM NaCl. Among the differentNaCl concentrations applied, 30mM NaCl did notsignificantly reduce the productivity of the ex-plants, and produced significant increases of thefresh and dry weights with respect to 0mM NaCl.Similar results have been observed in other woodyspecies (Singh et al., 2000; Sotiropoulos andDimassi, 2004). Flowers and Lauchli (1983) re-ported that, at low concentrations, NaCl exerts asignificant positive effect on shoot proliferation invitro due to the increased osmolarity.

With respect to visible symptoms of salt injuryobserved in the shoots, necrotic and fallen leaveswere appreciated from 60mM NaCl and apicalnecrosis from 120mM NaCl (Figure 1).

Salinity also significantly affected the rootinggrowth parameters (Po0.001 for all parameters,except for the fresh and dry weights of the shoot,P40.05). The growth parameters decreased

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Table 1. Effect of different NaCl concentrations on the growth parameters of Citrus macrophylla explants in theproliferation experiment.

NaCl (mM) Shoot number Shoot length(mm)

Productivity(mm)

Fresh weight/explant (mg)

Dry weight/explant (mg)

0 1.96a 20.1b 36.8a 41.1b 7.2b30 1.39bc 24.7a 31.8a 51.9a 8.5a60 1.87ab 13.7c 24.4b 38.2b 5.9c90 1.27c 15.8c 15.8b 35.4b 5.6c120 1.25c 11.7c 14.3c 25.7c 4.0d150 1.04c 12.5c 12.9c 15.0d 3.0d

Means followed by the same letter within each column are not significantly different at the 0.05 level, according to an LSD test.

Figure 1. Citrus macrophylla explants cultured in the proliferation experiment with different NaCl concentrations.A—0mM NaCl; B—30mM NaCl; C—60mM NaCl; D—90mM NaCl; E—120mM NaCl; and F—150mM NaCl.

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significantly from 40mM NaCl and were lowest at 80or 100mM NaCl (Table 2 and Figure 2). The rootingpercentage decreased from 87% at 0mM NaCl to30% at 100mM NaCl (Figure 2). Similar results wereobserved in grapevine, in which NaCl concentra-tions higher than 85mM prevented rooting(Troncoso et al., 1999). However, in sour orange,explants from seed yielded 80% rooting at 0, 50 or100mM NaCl, and no rooting occurred whenexplants were grown with NaCl Z150mM (Shiyabet al., 2003). The root number and length perexplant also decreased significantly with salinity: at100mM NaCl, these parameters were approxi-mately 45% lower compared with 0mM NaCl.

As in the proliferation experiment, visible symp-toms of salt injury in the shoots were observed(Figure 3). Treatment with 40mM NaCl produceddamage in 10% of leaves, whereas fallen leaveswere also observed from 60mM NaCl. At 100mM

NaCl, 68% of shoots had necrotic leaves, and 20%had fallen leaves (data not shown).

Mercado et al. (2000) observed that rooting wasthe parameter most affected by salt in tomato, asalso seen in potato (Martınez et al., 1996). Theseresults are in agreement with those observed inCitrus macrophylla; the salt effect on the growthparameters for shoots in proliferation was observedfrom 60mM NaCl, and in the rooting growth assayfrom 40mM NaCl.

Effect of NaCl on the mineral content ofexplants

The Na+ and Cl� concentrations in the ‘Macro-phylla’ explants increased significantly with thesalinity level in both the proliferated (Po0.001 forboth ions) and rooted (Po0.001 for both ions)explants (Figure 4). This pattern, the result ofincreasing NaCl in the medium, is a common andexpected in vitro response and has been reportedfor many species (Sotiropoulos, 2007; Sotiropoulosand Dimassi, 2004). For plants with a poor ability toexclude saline ions, marked injury in older leavescan occur within days (Munns, 2002). These saltinjuries could be due to accumulation of Na+ or Cl�

(or both) to excessive levels in transpiring leaves,building up rapidly in the cytoplasm and inhibitingenzyme activity, or in the cell walls, altering theirfunctioning (Flowers and Yeo, 1986). In citruscultivated in vivo, chloride accumulation is theprincipal cause of salt toxicity (Moya et al., 2003).In ‘Macrophylla’ explants in proliferation, chlorideincreased more than sodium; Cl� was 30 timeshigher at 150mM NaCl with respect to 0mM NaCl,whereas Na+ was only 8 times higher (Figure 4).These results suggest that the important deleter-ious effects in the Citrus macrophylla explantsgrown in vitro at increasing NaCl concentration

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Table 2. Effect of different NaCl concentrations on the growth parameters of Citrus macrophylla explants in therooting experiment.

NaCl (mM) Shoot length(mm)

Rootnumber

Root length(mm)

Fresh weight/explant (mg) Dry weight/explant (mg)

Shoot Roota Shoot Roota

0 28.81a 3.19a 25.80a 86.0 62.0a 18.5 9.1a20 30.26a 3.05ab 23.57ab 99.9 55.4ab 20.4 8.2ab40 26.35b 2.89ab 20.68bc 76.4 37.0bc 16.0 5.9bc60 25.38bc 2.48bc 17.48c 71.6 31.5bc 15.3 5.3bc80 24.27c 2.00c 18.62c 66.2 23.7c 13.8 4.1c100 24.23c 1.83c 13.53d 75.1 17.6c 15.9 3.0c

Means followed by the same letter within each column are not significantly different at the 0.05 level, according to an LSD test.aWeight per rooted explant.

NaCl (mM)

0

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0

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100a a

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Figure 2. Rooting percentage of Citrus macrophyllaexplants cultured in vitro with different NaCl concentra-tions. Vertical bars are standard errors. Bars with thesame letter are not significantly different at the 0.05level according to an LSD test.

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could be due to toxic intracellular levels of salineions, mainly Cl�. Other in vivo studies have reportedthat the lemon cultivar ‘Fino 49’ on ‘Macrophylla’accumulated less Na+ in its leaves than on ‘SourOrange’, indicating a capacity of Citrus macrophyllarootstocks for exclusion of Na+ (Garcıa-Lidon et al.,

1997). Although the Na+ levels were similar, theCl� concentrations in the proliferated explantswere greater than those of the rooted explants,since the root system, which controls ion exclusion,was absent and the shoots could directly absorb theions present in the medium.

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Figure 4. Effect of different NaCl concentrations on the Na+ and Cl� concentrations of Citrus macrophylla explantscultured in the proliferation or rooting experiment. Vertical bars are standard errors. Bars with the same letter are notsignificantly different at the 0.05 level according to an LSD test.

Figure 3. Citrus macrophylla explants cultured in the rooting experiment with different NaCl concentrations. A—0mMNaCl; B—20mM NaCl; C—40mM NaCl; D—60mM NaCl; E—80mM NaCl; and F—100mM NaCl.

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The Ca2+, K+, Mg2+ and Fe concentrations inplants were also affected by the NaCl concentra-tion of the medium. In the proliferated explants,the rise in NaCl significantly increased the concen-trations of these ions (Po0.001 for all ions; Table 3).These results are in disagreement with thoseobserved by Sotiropoulos and Dimassi (2004) inkiwifruit; Fe concentrations were not significantlyaffected, whereas K+ and Ca2+ decreased in thepresence of NaCl. For in vitro plants in prolifera-tion, the mechanisms implicated in mineral uptakeare different from those used by plants growing exvitro. Thus, mineral nutrients could be taken updirectly from the culture medium, mainly via thecut area, as demonstrated previously for IAA (Guanand De Klerk, 2000). The linear relationshipsbetween concentrations of some nutrients in thegrowth medium and in the plant tissues suggestuptake by passive diffusion. However, modificationof mineral concentrations in the culture medium donot always result in similar changes in plant tissues,suggesting that other mechanisms are also likelyinvolved in regulating mineral concentrations intissues (Gribble et al., 2002). There are interac-tions between minerals in the medium and/orbetween minerals and the agar matrix that influ-ence mineral availability and uptake. In vitro,mineral uptake is proportional to the mineralavailability, which is influenced by mineral diffu-sion—the dominant process in mineral availability—so the diffusion rate of minerals through the gelled

medium is an important factor in their uptake rateand tissue concentrations (Amiri, 2001). Theanomalous results found in proliferated explants,lacking a root system, could be related to differ-ences in the diffusion rates of mineral nutrients inthe culture medium with high NaCl concentrations.

In the rooting experiment, although the Fe, Mg2+

and K+ concentrations were not affected signifi-cantly (P40.05) by the salinity, NaCl significantlyreduced the Ca2+ concentration (Po0.05; Table 3).In vivo, plants take up mineral nutrients via theirroots, and the presence of NaCl in the mediumreduced the K+, Ca2+, Mg2+ and Fe concentrations incitrus leaves (Banuls et al., 1990).

Salinity significantly reduced the NO3� concentra-

tion in the explants, both in proliferation and inrooting experiments (Po0.001; Table 3). With thehighest NaCl concentration, the NO3

� concentrationwas decreased by 63% in proliferated and rootedexplants. In the rooting experiment, this wasprobably due to an antagonistic effect of Cl� onNO3� uptake, as has been described in plants ex

vitro (Grattan and Grieve, 1999). Cerezo et al.(1997) showed that Cl� accumulation in citrustissues reduced NO3

� concentrations by 70–80%. Inproliferated explants, the root system is absent, sono interactions between Cl� and NO3

� uptake couldexplain the lower NO3

� concentrations found inthese explants. Since the uptake of mineralnutrients is dependent on their diffusion throughthe medium (Amiri, 2001), it is possible that thediffusion rate of NO3

� decreases with increasingosmotic potential of the medium due to salinity,resulting in a lower NO3

� availability.

Effect of NaCl on physiological parameters

It is well established that the rate of photosynth-esis diminishes with stress, accompanied by thedegradation of chlorophyll and chlorophyll–proteincomplexes (Srivastava et al., 1988). In our study,the total chlorophyll concentration in the prolifer-ated explants was decreased significantly by NaCl(Po0.001), except at 30mM (Figure 5), and theexplants exhibited a highly chlorotic appearancefrom 60mM NaCl (Figure 1). This effect was morepronounced (Po0.001) in the rooted explantscultured with different concentrations of NaCl(Figure 5). The total chlorophyll concentration inexplants rooting in 100mM NaCl was decreased by62% relative to 0mM NaCl, and chlorotic leaveswere observed from 40mM NaCl (Figure 3). Inproliferation, the chlorophyll concentration was32% lower at 150mM NaCl than at 0mM NaCl. Inother woody species, high concentrations of NaCl

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Table 3. Effect of different NaCl concentrations on theCa2+, K+, Mg2+, Fe and NO3

- concentrations (mmol kg�1 DW)of Citrus macrophylla explants cultured in the prolifera-tion and rooting experiments.

NaCl (mM) Ca2+ K+ Mg2+ Fe NO3�

Explants cultured in the proliferation experiment0 111.5c 611.1b 59.8b 4.8b 436.1a30 139.4b 607.3b 68.7b 5.9b 259.6bc60 174.4a 793.7a 97.3a 9.4a 215.0cd90 158.9ab 770.4a 95.3a 9.3a 194.7cd120 153.9ab 784.8a 102.0a 8.9a 197.6cd150 157.0ab 748.2a 89.9a 9.3a 160.2d

Explants cultured in the rooting experiment0 230.8a 506.0 73.9 3.2 52.4a20 187.8b 540.9 80.2 4.2 45.5a40 183.9b 602.0 75.0 3.8 44.7a60 184.8b 636.5 73.9 4.3 23.2b80 164.8b 579.7 70.4 3.7 25.5b100 189.7b 618.8 74.4 4.3 19.1b

Means followed by the same letter within each column are notsignificantly different at the 0.05 level, according to an LSD test.

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applied under in vitro conditions also reduced thechlorophyll content (Chatzissavvidis et al., 2008;Erturk et al,. 2007).

The accumulation of activated oxygen speciesleads to lipid radical formation and, subsequently,cell membrane breakdown as well as other severedamage. Thus, the estimation of MDA, which is asecondary end-product of polyunsaturated fatty acidoxidation, is used widely to measure the extent oflipid peroxidation as an indicator of oxidative stress(Arbona et al., 2003) In explants of Citrus macro-

phylla in proliferation, the concentration of MDAincreased significantly from 30mM NaCl (Figure 6).The highest salinity level increased MDA by 40%relative to 0mM NaCl, indicating an important degreeof membrane damage. Queiros et al. (2007) obtainedsimilar results in cell lines of Solanum tuberosum, thehighest level of MDA occurring at 100–150mM NaCl.However, other studies of Carrizo citrange in vivo didnot find high levels of MDA at 90mM NaCl, sincethe antioxidant machinery was sufficient to avoidsignificant damage (Arbona et al., 2003).

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Figure 5. Effect of different NaCl concentrations on the total chlorophyll, proline and QAC concentrations of Citrusmacrophylla explants cultured in the proliferation or rooting medium. Vertical bars are standard errors. Bars with thesame letter are not significantly different at the 0.05 level according to an LSD test.

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To counter increasing external salinity, plantcells must adjust osmotically, either by accumulat-ing ions or by increasing their synthesis of organicsolutes such as sugars, proline and QAC (Fergusonand Grattan, 2005). In Citrus macrophylla explantsin proliferation, the increased K+ concentration(Table 3) may have been related to osmoticadjustment in the face of high external NaClconcentrations, although high concentrations ofother compatible solutes (proline and QAC) werealso observed (Figure 5). In addition to their rolesas osmoregulators, proline and QAC also could beinvolved in protection of cellular structures againstoxidative damage, by scavenging free radicals(Tsugane et al., 1999). Although Na+ and Cl�

accumulation also increased with salinity and couldhave contributed to cellular osmotic adjustment,the high accumulation of these specific ions couldhave been toxic and responsible for the verysignificant damage observed in the explants(Figures 1 and 3).

In the proliferation experiment, proline levels in‘Macrophylla’ explants increased significantly(Po0.001) from 60mM NaCl (Figure 5), risingin line with the external salt concentration.Important, positive correlations were found be-tween explant proline levels and saline ions in theproliferation experiment (Cl�: r ¼ 0.874, Po0.001and Na+: r ¼ 0.835, Po0.001). Similar results wereobserved in Populus callus, where even a lowconcentration of NaCl activated the mechanism ofproline accumulation (Zhang et al., 2004). How-ever, Citrus macrophylla explants in the rootingexperiment did not show significant differences in

their proline level as the salinity in the externalmedium increased (Figure 5).

In poplar, the QAC content increased withincreasing external NaCl concentration, being4 times higher than that of the control at 250mMNaCl (Zhang et al., 2004). We found similar resultsfor QAC in the Citrus macrophylla explants of theproliferation experiment. The increase in QACconcentration with salinity was highly significant(Po0.001), and was 150 times higher at 30mM NaClcompared with the control (Figure 5). The QACconcentration was also positively correlated withthe Cl� (r ¼ 0.943, Po0.001) and Na+ (r ¼ 0.828,Po0.001) concentrations in the proliferation ex-plants. Although in the rooting experiment ‘Macro-phylla’ explants at 100mM NaCl had 2 times moreQAC than the control explants, the differenceswere not significant (Figure 5).

For osmotic adjustment, proliferation and rootedexplants showed differing behaviors. Similar levelsof Na+ were found in the explants of the twoexperiments, but approximately 4 times more Cl�

accumulated in proliferated than in rooted ex-plants (Figure 4). In addition, proliferated explantsaccumulated proline and QAC under salinity,whereas rooted explants did not (Figure 5). Theaccumulation of proline and QAC in proliferatedexplants under external salinity indicates theimportant role of compatible solutes in the osmoticadjustment of the cytoplasm and its organelles. It islikely that proliferated explants employed vacuolarcompartmentation to avoid elevated levels ofcytoplasmic Cl� (Lloyd et al., 1989; Storey andWalker, 1999) and consequent damage to cytoplas-mic enzymes. If Cl� and other ions are sequesteredin the cell vacuole, K+ and other organic solutesshould accumulate in the cytoplasm and organellesto balance the osmotic pressure in the vacuole(Munns, 2002). In the proliferation experiment, K+,proline, and QAC accumulated significantly inexplants (Table 3 and Figure 5). Perhaps rootedexplants did not need to compartmentalize theirlow Cl� levels, and no accumulation of organicsolutes was required.

In summary, we investigated the capacity ofCitrus macrophylla explants, cultured in vitro, togrow under different NaCl concentrations. Thegrowth rate of ‘Macrophylla’ explants in bothproliferation and rooting experiments decreasedat high concentrations of salt. Proline and QACaccumulated only in proliferated explants undersaline conditions, whereas the chlorophyll concen-tration decreased in both cases. We suggest thatthe important deleterious effects in the in vitroexplants of Citrus macrophylla grown at elevatedNaCl concentrations are due mainly to cellular

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NaCl (mM)

0

MD

A (

mg

kg-1

DW

)

0.6

0.8

1.0

1.2

1.4

1.6

e

bc

dd

a

30 60 90 120 150

Figure 6. Effect of different NaCl concentrations on theMDA concentration of Citrus macrophylla explantscultured in the proliferation experiment. Vertical barsare standard errors. Bars with the same letter are notsignificantly different at the 0.05 level according to anLSD test.

Citrus macrophylla explants affected by NaCl stress in vitro 1931

Author's personal copy

toxicity of saline ions, mainly Cl�. The results ofthis study illustrate the potential of using tissueculture for evaluation of citrus salt tolerance, sinceresponses are relatively fast, the generation timesare short, and the environment is controlled. Thephysiological and nutritional responses found in thisstudy could inspire further investigations to definethe correlation between the in vitro and ex vitrobehaviors of citrus under saline conditions. There isevidence that in vitro nodal segments of Citrusmacrophylla respond to salinity in a similar way tothe whole plant, so this technique could be used forpre-selection and evaluation of salt tolerance. Inaddition, further research is needed to investigatethe mechanisms involved in mineral uptake andtranslocation by in vitro explants grown undersaline conditions.

Acknowledgements

The authors thank Mrs. Silvia Andujar and BeatrizGarcıa for technical assistance in the laboratorywork. We also thank Dr. David Walker for the Englishcorrection. This work was supported by theInstituto Nacional de Investigacion Agraria, throughthe project RTA2007-00094-00-00, and the Eur-opean Social Fund.

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