Plant Cell, Tissue and Organ Culture 78: 209–216, 2004.© 2004 Kluwer Academic Publishers. Printed in the Netherlands.

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The effect of ventilation on in vitro response of seedlingsof the cultivated tomato and its wild salt-tolerant relativeLycopersicon pennellii to salt stress

David Mills1,∗ & Moshe Tal21Institutes for Applied Research, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel;2Department of Life Sciences, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel(∗requests for offprints: Fax: +972-8-6472984; E-mail: mills@bgumail.bgu.ac.il)

Received 18 June 2003; accepted in revised form 10 December 2003

Key words: hyperhydricity, salt stress, salt tolerance, tomato species, ventilation, vitrification

Abstract

Organs or plants grown in vitro do not always exhibit the same responses to salinity as the whole plant of same spe-cies grown ex vitro. The response to salinity (100 mM NaCl) of seedlings of the wild tomato species Lycopersiconpennellii acc. Atico (Lpa) and of the cultivated tomato L. esculentum cv. M82 (Lem), the former is known as salttolerant and the second as relatively salt sensitive under ex vitro conditions, was compared under in vitro conditionswith three different ventilation regimes. It was found that under salinity shoots of the wild species accumulated thesame or even more dry biomass than the control (roots somewhat less) under all ventilation levels. Growth of shootsand roots of the cultivated species was inhibited under the same conditions especially under the high ventilation.Ventilation reduced some abnormalities of leaf development related to hyperhydricity and consequently ventilatedleaves exhibited a more compounded structure, increased area, increased resistance to water loss and stomatafunctioning. Ventilation increased K+, Na+ and Cl− accumulation in shoots of both tomato species. This wasmore pronounced under salinity and in Lpa. This work indicates that differences that characterize whole plantsof these species in response to salinity under ex vitro conditions are exhibited also in whole plants grown in vitrounder high ventilation. It is suggested that ventilation is needed to evaluate well the response of whole plants tosalt stress applied in vitro.

Abbreviations: Lem – Lycopersicon esculentum cv. M82; Lpa – L. pennellii acc. Atico

Introduction

Cell or tissue in vitro cultures have been used for se-lecting for stress resistance and to examine for theexistence of a correlation between the mechanismsoperating in cultured cells and in cells of the wholeplant (Tal, 1990). Tal (1990) listed all possible cor-relations between the responses to salt of the wholeplant and those of tissues and cells developed from thatplant. A positive correlation, where both the wholeplant and cultured cells are tolerant or sensitive tosalt was interpreted, respectively, as an indication forthe operation or lack of cellular mechanisms of salttolerance in the whole plant. A negative correlationwhere the whole plant is salt tolerant but the iso-

lated cells are sensitive, was regarded as an indica-tion for mechanisms that depend for their operationon the organization of the cells in tissues or organs inthe whole plant. Another negative correlation, wherethe plant is sensitive to salt but the isolated cells aretolerant, was interpreted as due to the differences inenvironment surrounding the cells in culture and inthe leaf tissue (Gale and Boll, 1978; Flowers et al.,1985). Although effective salt exclusion at the cellularlevel can be advantageous in vitro (where carbohydratesupply should be unlimited) it can be disadvanta-geous for leaf cells in vivo for which the carbon sup-ply is not unlimited and ions excluded from leaf cellsand accumulated in the apoplast may induce waterdeficit and loss of turgor (Oertli, 1968). This case

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was suggested as an example for the possible ef-fects of the components of the medium on the cellresponse to salt and as a warning against potentialmistakes while interpreting the results of experimentsperformed in cell or tissue culture and especially whentrying to extrapolate from cultured cells to wholeplants.

Calli prepared from roots, stems and leaves of thewild species L. pennellii and L. peruvianum, whichwere shown to be more salt tolerant than the cultivarunder greenhouse conditions (Dehan and Tal, 1978),grew better and accumulated more Na+ and less K+than calli of L. esculentum under salinity (Tal et al.,1978). According to these authors the fact that theresponse of cells and differentiating tissues to salin-ity matched the response of the whole plant suggeststhat cellular mechanisms operate in the intact plant.Similar results were obtained and a similar conclusionwas suggested in other cases (Von Hedenstrom andBreckle, 1974; Smith and McComb, 1981a, b; Warrenand Gould, 1982; Warren et al., 1985; Chandler et al.,1986).

Doubts have been expressed on the validity ofcomparisons of in vitro cultured cells and greenhouse-grown intact plants, since, in addition to the or-ganizational differences, the two grow in completelydifferent external environments (Dracup, 1991). Theexternal environments may differ, in addition totemperature and light conditions, in nutrients, hor-mones and the composition of the surrounding at-mosphere. The use of in vitro-grown intact plantsand cells in such comparisons seemed to be oneway of reducing or eliminating some of the differ-ences.

In the present study therefore, we comparedgrowth (biomass accumulation), accumulation of K+,Na+ and Cl− ions and water loss in young plants ofthe cultivated tomato and its wild salt-tolerant relativeL. pennellii, grown in vitro in control and salinizedmedia under restricted and improved ventilation. Asfar as we know there are no reports concerning theinteraction of ventilation and the responses to salinityin vitro.

The results demonstrate that unlike under restric-ted ventilation, where the differential response of thetwo species does not reflect the ex vitro situation, theexposure of the in vitro-grown seedlings to ventilationmade their response to salinity closer to the situationex vitro, where L. pennellii is more tolerant to salinitythan L. esculentum.

Materials and methods

Plant material and culture conditions

Seeds of the cultivated tomato Lycopersicon escu-lentum Mill cv. M82 (Lem) were disinfected by treat-ing them for 1 min in 1% hypochlorite. Seeds of thewild salt-tolerant species L. pennellii (Corr.) D’Arcyaccession Atico (Lpa) were treated for 10 min in 3%hypochlorite for both disinfection and digestion of theseed coat. After three washings in sterile deionizedwater, the seeds were germinated on solidified 0.8%Bacto-Agar (Difco Laboratories) with half-strengthMS medium (Murashige and Skoog, 1962). Afterradicle emergence seedlings were transferred to fullMS medium (including 3% sucrose) with or without100 mM NaCl. MS medium without added NaCl con-tained 0.22 mM Na+ and 6.2 mM Cl−. Magenta boxes(375 ml in volume) sealed with either non-ventedlids (control, restricted ventilation) or with membrane(0.3 µm NPS)-vented lids (Osmotek Ltd, Rehovot,Israel) were used in the experiments. Two membranesused were 10 or 16 mm in diameter (correspondingto an area of 79 and 201 mm2, respectively). Seed-lings were kept in the growth vessels for 25 daysin a growth room at a temperature of 25 ± 1 ◦C un-der 45–55µmol m−2 s−1 photon flux supplied by coolwhite fluorescent bulbs and a photoperiod of 16 hlight.

Growth parameters

Upon termination of the experiment the followingparameters were determined: number of leaves, shoot(stem and leaves) and root fresh and dry (70 ◦Cfor 48 h) weights using 8–16 seedlings per spe-cies per treatment. For mean leaf area determina-tion, stems were separated into stems and leavesthat were photocopied, the image was scanned, andarea was measured with the Image Analysis soft-ware NIH Image and divided by the number ofleaves.

Ion content

Ions were extracted from shoots and roots as pre-viously described (Mills, 1988); K+ and Na+ weredetermined using Inductive Coupled Plasma, Perkin-flammer Optima 3000 and chloride with a chlorido-meter (442-5000 Haake Buchler).

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Table 1. Dry weight (mg) of shoots and roots of seedlings of the cultivated tomato (Lem) and its wildsalt-tolerant relative L. pennellii (Lpa) grown in MS medium without (C) or with 100 mM NaCl and underthree ventilation regimes: Magenta boxes with either 0- (closed lid), 10- or 16-mm membrane in diameter

Ventilation Shoot dry weight Root dry weight(mm membrane diameter)

Lem Lpa Lem Lpa

C NaCl C NaCl C NaCl C NaCl

0 35.1a 22.6ay 2.4a 2.0ax 40.0a 11.1az 3.0a 2.8ax

10 34.6a 17.8az 2.6a 3.3ax 44.1a 4.63bz 3.7a 2.5ax

16 72.5b 12.4az 2.7a 3.7ax 50.0a 2.72bz 3.4a 2.9ax

Values are means of eight replicates per treatment. Means in each column followed by different letters aresignificantly different (p < 0.05) as determined by Fisher’s protected LSD test.x,y,z Means of both treatments for each species do not differ statistically (p > 0.05), differ statistically at0.01 <p < 0.001 or at p < 0.0001, respectively.

Water relation parameters

For water loss determination, seedlings were taken outof the boxes and shoots which were separated from theroots, were weighed immediately and after 60 min un-der room conditions (usually 23 ± 2 ◦C and 61 ± 3%RH). Water loss was calculated by dividing the loss ofweight after designated periods by the total shoot area(determined by scanning as explained above).

The width of the stomata aperture was determinedin 45 stomata, using two or three leaves per treatment,immediately after taking the seedlings out of the grow-ing vessels and 30 min after being exposed to ambientconditions (as for water loss determination). Stomatalaperture was determined by the impression methodusing Xantopren H, silicone based curing impressionmaterial (Heraeus, Germany) with Speedex, a uni-versal activator (Coltene, Switzerland) applied on theadaxial surface of the leaves to get the negative impres-sion and nail varnish applied on the hardened polymerto get the positive one. Aperture width was deter-mined with a microscope equipped with a micrometereyepiece.

Statistics

Usually three boxes, each containing four seedlings,were used per treatment. Eight seedlings were col-lected randomly for growth and water loss mea-surements (three of them were used for ion extraction).The other seedlings were used for stomata aperturemeasurements. The study of the effect of ventilation,on control and salt-treated plants of the two tomato

species, was repeated three times with similar results.Here we report the results of one replication. Datawere evaluated by analysis of variance (Fisher’s pro-tected LSD, probability of 5%) using Super ANOVA.

Results

Seedlings of both species responded positively to ven-tilation under control (no salt) conditions: their shootsaccumulated more biomass, 13% in Lpa and twice inLem, under high ventilation (16-mm membrane in dia-meter) as compared to seedlings grown under restric-ted ventilation (Table 1). In both species, ventilation

Figure 1. Mean leaf area of seedlings of the cultivated tomato(Lem) and its wild salt-tolerant relative L. pennellii (Lpa) grownin MS medium with or without 100 mM NaCl and under threeventilation regimes. Values are means of eight replicates pertreatment ± S.E. The same letters designate no significant statis-tical difference (p > 0.05) between different ventilation treatmentsfor each species and either control or salt treatment. ns, ∗,∗∗designate no statistical difference (p > 0.05), statistical differ-ence at 0.05 <p < 0.001, and at p < 0.001 between control and salttreatments for each ventilation, respectively.

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Figure 2. Seedlings of the cultivated tomato (Lem) grown in MS medium with or without 100 mM NaCl and under two ventilation regimes:tubes covered with Parafilm or with Sun Cap closure foil, 10-mm membrane in diameter.

did not affect root biomass production significantlyunder these conditions. Shoot biomass production ofsalt-treated Lem decreased, being 64, 51, and 17%of the control, respectively, under ventilation of 0, 10and 16-mm membranes in diameter. Biomass produc-tion in salt-treated Lpa, which was less affected byventilation, was higher than in the control under themedium and high ventilation treatments, being 127,137%, respectively (Table 1). Root growth decreasedin Lem more than the shoots by salinity and, similarlyto the shoot, it became more sensitive to salinity withthe increase in ventilation. Root growth decreased bysalinity also in Lpa. This decrease, however, was muchsmaller than that in Lem.

Seedlings of both species produced morphologi-cally degenerated leaves with small or without leaf-lets at all under control conditions with restrictedventilation. Ventilation under control improved leafletdevelopment, as reflected by the increase of mean leafarea (total seedling leaf area divided by the number ofleaves) in both species (Figure 1), in Lpa under bothintermediate and high ventilation treatments and inLem only under the higher ventilation treatment (Fig-ure 2, shown only for Lem). Unlike in Lem, ventila-tion under salinity, similarly to the control, increasedleaf area significantly in Lpa. Salinity improved leafletdevelopment in Lem under restricted and intermediateventilation and in Lpa under only restricted ventilation(Figure 2). In both species, leaves were morphologi-cally well developed under salinity, but due to thedecrease of their number their total area was reducedas compared to the control, especially under the highventilation treatment.

Ventilation increased K+ accumulation in shoots(stem and leaves together) of both tomato species un-

der control and saline conditions: the increase wasgreatest in Lpa under control conditions (Figure 3A).Ventilation increased also Na+ and Cl− in shoots ofboth species under salinity: the increase was muchmore pronounced in Lpa. An increased ion contentby ventilation was observed also in roots (Figure 3B).Ventilation increased K+ in the root under control con-ditions only in Lpa and under salinity in Lpa underintermediate and high ventilation and in Lem onlyunder the high ventilation. Ventilation increased Na+and Cl− in both species under salinity: the increasewas greater in Lpa under restricted and intermediateventilation. As opposed to Lem, ventilation increasedsomewhat Na+ and Cl− ions also in the shoots andmore so in the roots of Lpa plants grown in the con-trol MS medium (Murashige and Skoog, 1962) thatcontains these ions among its components.

In both species shoot water loss under ambientconditions was significantly lower in seedlings thatwere developed under ventilation as compared to thosegrown under restricted ventilation (Figure 4). Reduc-tion of water loss by ventilation was greater in Lpaunder control conditions and similar in the two speciesunder saline conditions. Salt increased the resistanceto water loss, in Lpa only under restricted ventila-tion and in Lem under all ventilation treatments. Thewidth of stomatal aperture was determined only un-der control conditions either in leaves just taken outof the growth vessel (0 min) or in those exposed toroom environment for 30 min (30 min) (Table 2). Inleaves developed under restricted ventilation, Lpa sto-mata were less open than those of Lem at 0 min andmuch more closed after 30 min exposure. In leavesdeveloped under the intermediate and high ventila-tion treatments stomatal apertures were significantly

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Figure 3. Concentration of K+, Na+ and Cl− in leaves (A) androots (B) of the seedlings of cultivated tomato (M82) and its wildsalt-tolerant relative L. pennellii (Lpa) grown in MS medium with orwithout 100 mM NaCl and under three ventilation regimes. Valuesare means of eight replicates per treatment ± S.E. The same lettersdesignate no significant statistical difference (p > 0.05) betweendifferent ventilation treatments for each ion and either control orsalt treatment.

smaller than those of the restricted ventilation in bothspecies; the apertures of Lpa stomata however weresmaller than those of Lem.

Figure 4. Water loss from seedlings of the cultivated tomato (Lem)and its wild salt-tolerant relative L. pennellii (Lpa) grown in MSmedium with or without 100 mM NaCl and under three ventilationregimes. Other details as in Figure 1.

Table 2. Effect of ventilation on stomatal opening (µm) in leavesof the two tomato species under control and salinity treatment,0 or 30 min after leaves were transferred from boxes to ambientconditions

Ventilation L. esculentum L. pennellii

(mm membrane diameter)

0 min 30 min 0 min 30 min

0 11.4a 6.0A 9.0a 0.2A

10 2.3b 0.5B 0.4b 0.2A

16 1.4b 0.9B 0.2b 0.04A

In each column means followed by the same letters do not signific-antly differ at p = 0.05.

Discussion

Comparisons between whole plants grown ex vitroand isolated cells or tissues grown in vitro are usuallyperformed to clarify whether a certain mechanism orresponse that operates at the level of the intact plantoperates also at the level of the cell. In such compari-sons, however, the differences between the ex vitro andin vitro environments are not considered in additionto the difference in the differentiation level (Dracup,1991). One approach to negate the vast differencesthat exist between the two environments is to growthe intact plants, similarly to the isolated cells, un-der in vitro conditions. A prerequisite for such anapproach is that the response or mechanism that char-acterizes the intact plant under ex vitro conditionsoperates also under in vitro conditions. Therefore,we examined whether the lower salt tolerance thatcharacterizes the Lem plants and the higher tolerancethat characterizes Lpa under ex vitro conditions isexpressed also when they are grown under in vitroconditions.

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While the in vitro growth response of highly ven-tilated Lem seedlings to NaCl was similar to theirex vitro response, that is, they responded as less salttolerant, their growth under restricted ventilation wasonly slightly reduced by salinity. The reasons forin vitro Lem plants being relatively tolerant to salin-ity under restricted ventilation and more susceptibleunder improved ventilation can be explained at leastpartially by the difference in the atmospheres of thetwo environments. The gaseous composition of theunventilated in vitro atmosphere is relatively richer inCO2, ethylene and water vapors as compared to a reg-ular atmosphere (Wardle et al., 1983; De Proft et al.,1985; Smith et al., 1990; Jackson et al., 1991). Thehigh levels of these gaseous components, particularlywater vapors, are considered as the major causes forabnormalities observed in organs and plantlets grownin vitro with low ventilation (Ziv, 1990; Debergh et al.,1992). These abnormalities may include the lack ofvascular bundles, lignin synthesis and polyphenolsand waxes, thin cell walls, malfunction of stomata,low differentiation of mesophyll cells that may affectplant–water and plant–ion relationships and thus theresponse of the plant to stress. Slower water movementinto and through the shoot due to the lack of vascularbundles as well as the lack of water potential differ-ence between the in vitro atmosphere and the shoot,may be the cause for the decreased accumulation ofsalt ion in Lem leaves (Figure 3A) and consequentlyfor the relative increase in salt tolerance under the re-stricted ventilation. Osmotic balance in these leavesmay be obtained by sucrose that is absorbed from themedium. It should be noted that the decrease in dryweight was due to inhibition in stem (hypocotyl andepicotyl) elongation (see Figure 2). This was not offsetby the increase in leaf size (Figure 1).

Increased ventilation of the growth vessels resul-ted in improvement of leaf development and shootbiomass in Lem plants only under control conditions.There are numerous reports on the positive effect ofventilation on growth of plantlets in vitro by lessen-ing the abnormalities related to hyperhydricity (e.g.,Ziv, 1990; Cournac et al., 1991; Debergh et al., 1992;Kanechi et al., 1998; Lai et al., 1998). Improvedvascular system may be the cause for an increase oftransport and accumulation of salt ions in Lem leaves(Figure 3A) and thus for the increase growth inhibitionby salt in these plants under the improved ventilation.Lem plants, in contrast to those of Lpa (Dehan andTal, 1978; Tal and Shannon, 1983), cannot cope withhigh concentrations of salt ions, since, most likely,

salt ions in leaves of the former are not compart-mentalized in the vacuole as in the latter (Taha et al.,2000).

Unlike in vitro seedlings of the cultivated species,those of the wild species Lpa responded as tolerant tosalt under either low or high ventilation, similarly totheir response to salt under ex vitro conditions. Growthof in vitro Lpa seedlings was not impaired when ex-posed to high salinity. They accumulated high levels(up to 300 mM) of Na+ and Cl− in the shoots androots, and lower K+ level in the shoot and resistedbetter water loss under ambient conditions (especiallywhen developed under restricted ventilation). UnlikeLem, in vitro Lpa seedlings exhibited salt tolerance,that is, growth unimpairment, under both restric-ted and improved ventilation in spite of the relativedecrease of salt ions accumulated under the formerconditions. The high accumulation of salt ions in leafcells of Lpa, as compared to Lem, under both restric-ted and improved ventilation and their probable bettercompartmentation within the vacuole may explain thehigher salt tolerance of Lpa under both conditions.

Deviation of the differential response of Lem andLpa plants to salinity under in vitro as compared tothe ex vitro conditions was reported also by Canoet al. (1998) who found that the difference betweensalinized plants of cultivated tomato and Lpa in theirgrowth under ex vitro conditions, was not clearlyshown on the basis of the growth of in vitro salin-ized plantlets. Similarly, Hanson (1984) reported thatshoot apices and roots of Lpa were not more tolerantto salinity in vitro than those of the cultivated tomato.Plantlets of Lpa grown in vitro under restricted venti-lation were more sensitive to 0.8% NaCl than thoseof the cultivated tomato (see Figure 19.1 in Hanson,1984). Though not indicating any abnormalities re-lated to hyperhydricity, restricted ventilation couldhave caused the lack of difference between the twospecies in both cases.

Prevention of shoot water loss under ambient con-ditions was somewhat improved in seedlings of Lemthat were developed under improved ventilation. Re-duction in ambient water loss in the in vitro plants de-veloped under improved ventilation could stem fromeither better stomata functionality and/or productionof more epicuticular waxes in the cuticule layer ofthe leaf. By scanning electron microscope we ob-served no crystals of epicuticular waxes on the surfaceof any of the leaves of tomato leaves grown in cul-ture as opposed to many other plants (e.g., jojoba,Mills et al., 1997). It is thus difficult to assess the

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contribution of epicuticular wax on water loss fromleaves of tomato. However, judging from stomatalaperture size (Table 2), it seems that under improvedventilation stomata are functioning well than under re-stricted ventilation. The values of the accumulationof salt ions, and stomatal aperture size in Lem un-der in vitro conditions became more similar to thoseof ex vitro conditions when ventilation increased. Inaddition, in both species, increase in ventilation res-ulted in a decrease in succulence (FW/DW) (data notshown), reaching similar values reported for the samespecies grown ex vitro (Dehan and Tal, 1978; Taland Shannon, 1983). Succulence was shown to de-crease with the increase in ventilation also in otherspecies (Ziv, 1990; Majada et al., 1997; Mills et al.,2001).

The effect of salinity stress is perhaps equivalent tothe effect of osmotic stress caused by elevated levelsof sucrose, fructose and mannitol or by increasedlevels of gelling agents that decreased water poten-tial, both resulting in the reduction of hyperhydricityas reported by Debergh (1983) and Ziv (1990) NaClexhibited positive effects on the plants of both speciesgrown in vitro under restricted ventilation that are ex-pressed by improved leaflet development (Figures 1and 2) and increased resistance to water loss (probablyby reducing stomata aperture) as determined underambient conditions. In an earlier study (Mills andBenzioni, 1992), salinity was reported to stimulate leafgrowth and wax deposition on leaf surface of leaves ofjojoba seedlings grown in vitro and thus reduced waterloss under ambient conditions (Mills et al., 1997). Intheir study on in vitro-grown plants of L. esculentum,Bourgeais-Chaillou and Guerrier (1992) reported thatshoots produced about 30% more fresh biomass un-der 25–50 mM NaCl than under the control. Apices ofthe cultivated species had developed to much healthiernon-epinastic plantlets in 0.8% NaCl as compared toplantlets of the control (see Figure 19.1 in Hanson,1984).

In conclusion, it is suggested that ventilation is ne-cessary when the response of whole plants and eithercells, tissue or organs are compared under in vitro con-ditions. The current study suggests that due to someabnormalities related to hyperhydricity, the responseof especially the salt-sensitive cultivated tomato to sa-linity under in vitro conditions with restricted venti-lation is not as expected from ex vitro experiments, andthat the response under in vitro conditions resemblesthe one under ex vitro conditions only when ventilationis applied.

Acknowledgements

The authors thank Ms Shivta Wenkart for her valu-able technical support. We also thank the Dr HermanKessel Fund, which is dedicated to the memory ofMr C.J.J. van Rensburg, for its partial support of thisstudy.

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