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Environmental and Experimental Botany 60 (2007) 397–403

Improved salt tolerance of melon (Cucumis melo L.) bythe addition of proline and potassium nitrate

Cengiz Kaya a,∗, A. Levent Tuna b, Muhammad Ashraf c, Hakan Altunlu d

a Harran University, Agriculture Faculty, Soil Science and Plant Nutrition Department, Sanliurfa, Turkeyb Mugla University, Biology Department, Mugla, Turkey

c Department of Botany, University of Agriculture, Faisalabad 38040, Pakistand Mugla University Ortaca Polytechnic, Mugla, Turkey

Received 12 April 2006; received in revised form 1 December 2006; accepted 28 December 2006

bstract

A pot experiment was carried out under glasshouse conditions with melon (Cucumis melo) cv. “Tempo F1” in a mixture of peat, perlite andand (1:1:1) to investigate the effects of external proline and potassium nitrate applications to salinity-treated (150 mM) plants with respecto fruit yield, plant growth, some physiological parameters and ion uptake. Treatments were—(i) control (C): plants receiving nutrient solu-ion, (ii) salinity treatment, as for control plus 150 mM NaCl. Salinity treatment was combined with or without either 5 mM supplementaryNO3 or 10 mM proline. The salt treatment (150 mM NaCl) led to significant decreases in plant growth, fruit yield, relative water content

RWC), stomatal density, uptake of Ca2+, K+ and N, and chlorophyll a and b contents, accompanied by significant increases in Na+ uptake,roline concentration and membrane permeability. Supplementary KNO3 and proline treatments significantly ameliorated the adverse effects

f salinity on plant growth, fruit yield and the physiological parameters examined. This could be attributed to the effects of all the externalupplements in maintaining membrane permeability, and increasing concentrations of Ca2+, N and K+ in the leaves of plants subjected to salttress.

2007 Elsevier B.V. All rights reserved.

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eywords: Amelioration; Exogenous application; Salt tolerance; Fruit; Salinity

. Introduction

Salinity is a major abiotic stress reducing the yield of aide variety of crops all over the world (Tester and Davenport,003; Ashraf and Foolad, 2007). Although the level of saltsn most irrigation waters is below the threshold for the moreensitive crops, salt accumulation in irrigated soils from bothrrigation and groundwater sources can increase salinity to lev-ls which can reduce growth and yield of even the more tolerantrops. Overcoming salt stress is a main issue in these regions tonsure agricultural sustainability and continued food productionHeuer, 2003).

Sodium is the predominant soluble cation in many of theoils of arid and semi-arid areas. However, most plants, espe-ially glycophytes, are very sensitive to high Na+ concentrations.

∗ Corresponding author. Tel.: +90 4142470384/2309; fax: +90 4142474480.E-mail address: c kaya70@yahoo.com (C. Kaya).

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098-8472/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2006.12.008

ine; Potassium nitrate

igh concentrations of Na+ disturb intracellular ion homeostasishich leads to membrane dysfunction, attenuation of metabolic

ctivity, and secondary effects that cause growth inhibitionhereby leading to cell death (Rus et al., 2001, 2004; Ashraf,004). The ability of plant cells to maintain sodium concentra-ions low in the cytosol is a vital process associated with thebility of plants to grow under high salt regimes (Blumwald,000; Ashraf and Harris, 2004). Sodium enters leaf cells and ishen pumped into the vacuole before its concentrations are builtp in the cytoplasm. The pumping of Na into the vacuole is cat-lyzed by vacuolar Na+/H+ antiporter (Blumwald et al., 2000)nd the plasma membrane Na+ transporter AtHKT1 (Rus et al.,004). However, Lacan and Durand (1996) proposed that underigh Na+ concentration, a Na+/H+ antiporter worked in reverse,o pump Na+ into the cytosol.

Plants need to maintain internal water potential below thatf soil to maintain turgor and sufficient water for growth. Thisequires an increase in osmotica, either by uptake of solutesrom the soil or by synthesis of metabolically compatible solutes

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Tester and Davenport, 2003). Compatible solutes, such as pro-ine, are known to accumulate under conditions of environmentaltresses to play a role in the process of osmotic adjustmentn many crops (Heuer, 1994). Their main role is probably torotect plant cells against the ravages of salt by preservinghe osmotic balance, stabilizing sub-cellular structures, suchs membranes and proteins, and scavenging reactive oxygenpecies (Heuer, 2003; Ashraf and Foolad, 2007). Therefore,xogenous application of these compounds has been suggesteds an alternative or additional approach to genetic engineeringo improve crop productivity under stress conditions (Makale etl., 1996).

Potassium is a major plant macro-nutrient that plays impor-ant roles related to stomatal behavior, osmoregulation, enzymectivity, cell expansion, neutralization of nondiffusible nega-ively charged ions and membrane polarization (Maathuis andanders, 1996; Elumalai et al., 2002). Metabolic toxicity ofa+ is largely due to its ability to compete with K for binding

ite essential for cellular function (Bhandal and Malik, 1988).hus, high levels of Na or high Na:K ratios can disrupt vari-us enzymatic reactions in the cytoplasm (Blaha et al., 2000).ith high concentrations of Na+ in the leaf apoplast and/or vac-

ole, plant cells have difficulty in maintaining low cytosolica+ and, perhaps as importantly, low Na+:K+ ratios (Gorham et

l., 1990; Maathuis and Amtmann, 1999). In fact, high K+:Na+

atio is more important for many species than simply main-aining a low concentration of Na+ (Cuin et al., 2003). High

+/Na+ ratios are also closely related to salt resistance in arti-hoke (Graifenberg et al., 1995), tomato (Lopez and Satti, 1996),hickpea (Ozcan et al., 2000) and Brassica napus (Qasim andshraf, 2006).It is evident that salt stress has a significant effect on N nutri-

ion in plants. Salinity reduces the uptake of NO3− in many plant

pecies mostly due to high Cl− content of saline soil (Grattannd Grieve, 1994; Khan and Srivastava, 1998). However, sup-lementing soil with N improved plant growth under salt stressDubey and Pessarakli, 1995).

Attempts to improve tolerance to salinity through con-entional plant breeding methods are time-consuming andaborious, and rely on existing genetic variability. Moreover,t is difficult to modify single traits that are probably multi-ene controlled. Taking advantage of the substantial increasef knowledge about physiological responses, the use of physi-logical selection criteria can improve the success of empiricalelection for agronomic traits (Noble and Rogers, 1992).

To overcome the negative impact of high salinity on a crop, itould be advantageous to add supplemental proline and KNO3o a growth medium that is known to be, or may become salinet some time during the crop growth cycle. An experiment wasonducted with melon plants in pots to assess the effectivenessf supplemental proline or KNO3 to mitigate the adverse effectsf salinity stress. The aim was to determine if this would correct

and N deficiencies in the presence of high NaCl and also

o assess effects of supplemental K, N and proline on someey growth and physiological parameters (e.g. dry weight, fruitield, relative water content, membrane permeability and prolineontent).

w6

imental Botany 60 (2007) 397–403

. Materials and methods

.1. Plant culture and treatments

The experiment was conducted under greenhouse conditionsn Mugla-Ortaca (Turkey) from April to the end of June 2003ith melon (Cucumis melo L.) cv. Tempo F1. The relativeumidity was kept between 50 and 60%. Three seeds of melonere sown directly in plastic pots containing 8.0 kg of peat, per-

ite and sand mixture in equal ratios; following germination,lants were thinned to one plant per pot.

The pots were covered with black plastic sheet to preventvaporation. The composition of the nutrient solution used wasin mM): 15 N (in NO3 form), 1 P, 6 K, 5 Ca, 2 Mg, 2 S, 0.05e, 0.01 Mn, 0.05 B, 0.003 Cu, 0.0008 Zn and 0.001 Mo.

Twenty days after germination different treatments were initi-ted. Treatments were—(i) control (C): plants receiving nutrientolution, (ii) salinity treatment (C + S): plants receiving nutrientolution plus 150 mM NaCl, (iii) salinity plus supplementarymM KNO3 (C + S + KN): plants receiving nutrient solutionnd 150 mM sodium chloride (NaCl) plus 5 mM supplemen-ary KNO3, and (iv) salinity plus supplementary 10 mM prolineC + S + Pro): plants receiving nutrient solution and 150 mMaCl plus 10 mM supplementary proline applied foliarly twiceweek. The volume of proline spray pulverized ranged from 25

o 50 ml per pot depending on plant age. The same amount ofater was pulverized to the control plants. Each treatment was

eplicated three times and each replicate included 5 pots (i.e. 15ots per treatment). The pH of the nutrient solution was adjustedo 6.0 with 0.1 mM KOH during the entire growing period. Theolume of the nutrient solution applied to the root zone of thelants ranged from 200 to 500 ml from April to June each timeswice a week depending on plant age. Plants were harvested 90ays after seedling emergence.

.2. Fruit yield

The values for the total fresh fruit weight per plant are theeans of the fruit yield of three plants per replicate and presented

n kilograms per plant.

.3. Chlorophyll determination

Prior to extraction, fresh leaf samples were cleaned witheionized water to remove any surface contamination. Chloro-hyll extraction was carried out on fresh fully expanded leafaterial; one g leaf sample was ground in 90% acetone usingpestle and mortar. The absorbance was measured with aV/visible spectrophotometer (Pye Unicam SP6-550, UK) and

hlorophyll concentrations were calculated using the equationroposed by Strain and Svec (1966).

Chl b (mg ml−1) = 20.97 × (A645) − 3.94 × (A663)

here (A663) and (A645) represent absorbance values read at63 and 645 nm wavelengths, respectively.

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.4. Electrolyte leakage

This parameter was included in order to have more infor-ation on membrane stability and thereby on the relative ion

ontent in the apoplastic space. Electrolyte leakage was assesseds described by Lutts et al. (1996) using nine young leaf discs forach treatment. Samples were washed three times with deionizedater to remove surface-adhered electrolytes. Leaf discs werelaced in closed vials containing 10 ml of deionized water andncubated at 25 ◦C on a rotary shaker for 24 h; subsequently elec-rical conductivity of the solution (Lt) was determined. Samplesere then autoclaved at 120 ◦C for 20 min and the last electrical

onductivity (L0) was obtained after equilibration at 25 ◦C. Thelectrolyte leakage was defined as follows:

lectrolyte leakage (%) =(

Lt

L0

)× 100.

.5. Proline determination

Proline was determined according to the method describedy Bates et al. (1973). Approximately, 0.5 g of fresh leaf mate-ial was homogenized in 10 ml of 3% aqueous sulfosalicylic acidnd filtered through Whatman’s No. 2 filter paper. Two milliliterf the filtrate was mixed with 2 ml of acid-ninhydrin and 2 mlf glacial acetic acid in a test tube. The mixture was placed in aater bath for 1 h at 100 ◦C. The reaction mixture was extractedith 4 ml toluene and the chromophore containing toluene was

spirated, cooled to room temperature, and the absorbance waseasured at 520 nm with a Shimadzu UV 1601 Spectropho-

ometer. Appropriate proline standards were included for thealculation of proline in the samples.

.6. Leaf relative water content

Leaf relative water content (LRWC) was calculated based onhe methods from Yamasaki and Dillenburg (1999). Two leavesrom two randomly chosen plants per replicate were collectedrom the mid-part of the plants in order to minimize age effects.ndividual leaves were first removed from the stem and theneighed to obtain fresh mass (FM). In order to determine the

urgid mass (TM), leaves were floated on distilled water inside alosed petri dish. Maximum turgidity was determined by weigh-

ng leaves (after gently wiping the water from the leaf surfaceith tissue paper) until no further weight increase occurred. At

he end of the imbibition period, leaf samples were placed in are-heated oven at 80 ◦C for 48 h, in order to obtain dry mass

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able 1ffects of NaCl and NaCl plus potassium nitrate or proline on dry weight (g/plant),ixture of peat, perlite and sand and irrigated with a standard nutrient solution

reatments Shoot DW Root DW Whole plant DW

18.38a 9.22a 27.60a+ S 13.74b 7.91b 21.65b+ S + KN 17.39a 9.15a 26.54a+ S + Pro 16.42a 9.57a 25.97a

ithin each column, same letters (a–c) indicate no significant difference among tre50 mM sodium chloride added to nutrient solution; KN: 5 mM KNO3 supplemented

imental Botany 60 (2007) 397–403 399

DM). All mass measurements were made using an analyticalcale, with a precision of 0.0001 g. Values of FM, TM and DMere used to calculate LRWC using the equation below:

RWC (%) =[

FM − DM

TM − DM

]× 100

.7. Stomatal density

Stomatal density was determined on the same leaves usedor relative water content. Leaf impressions were obtained fromoth surfaces of the basal leaflet of five leaves per cultivar and saltreatment. Impressions were examined with a light microscopet 400× magnification, and stomata were counted on 10 fieldsaken at random from each sample.

.8. Dry weight determinations and chemical analysis

Three randomly selected plants per replicate were collectedt the fruit set stage, divided into leaves, stems and roots, andried in an oven at 70 ◦C for 2 days to determine dry weightsnd elemental concentrations. Chemical analyses were carriedut on a dry weight basis. Total N was determined in samples of.0 g dry weight using a Kjeldahl method. Ground samples werery-ashed at 550 ◦C for 4 h, mixed with hot 2 M HCl, filtered,nd then brought to a final volume of 50 ml with distilled water.alcium (Ca), K and Na were determined in these digests usingn Eppendorf flame photometer (Chapman and Pratt, 1982).

.9. Statistics

The data for all parameters were statistically analyzed to workut two-way analysis of variance using the Statview-ANOVAomputer package. Actual data were used for computingNOVAs for each attribute except electrolyte leakage and

eaf relative water content (RWC) for which actual data wereransformed into percentages before ANOVA was applied. Sta-istically different groups were compared using an LSD testP < 0.05).

. Results

The results obtained from this experiment show that highalinity reduced fruit yield, plant dry matter, chlorophyll con-ents, number of stomata and the relative water content of leavesTables 1 and 2). However, both externally supplied chemicals

fruit weight (kg/plant) and chlorophyll content (mg/kg), for melon grown in

Fruit weight Chl a Chl b Total Chl

6.30a 890b 500b 1390b4.05c 660c 420c 1080c5.24b 1030a 550a 1580a5.36 b 990ba 520a 1510a

atments at the P < 0.05 level. C, control: complete nutrient solution alone; S:in nutrient solution and Pro: 10 mM proline applied foliarly.

400 C. Kaya et al. / Environmental and Experimental Botany 60 (2007) 397–403

Table 2Effects of NaCl and NaCl plus potassium nitrate or proline on electrolyte leakage (%), proline content (�moles/g FW), relative water content (%) and stomataldensity (units/mm2) for melon grown in mixture of peat, perlite and sand and irrigated with a standard nutrient solution

Treatments Electrolyte leakage Proline content Relative water content Stomatal density

C 11.5c 0.41d 78.32a 175aC + S 42.7a 0.92b 59.41c 125dC + S + KN 25.3b 0.74c 70.76b 147bC + S + Pro 14.4c 2.02a 76.75a 138c

Within each column, same letter (a–c) indicates no significant difference among treatments at the P < 0.05 level. C, control: complete nutrient solution alone; S:150 mM sodium chloride added to nutrient solution; KN: 5 mM KNO3 supplemented in nutrient solution and Pro: 10 mM proline applied foliarly.

Table 3Effects of NaCl and NaCl plus potassium nitrate or proline on Na, K, Ca and N concentrations (% dry matter) in the leaves and roots of melon grown in a mixture ofpeat, perlite and sand and irrigated with a standard nutrient solution

Treatments Leaf Root

Na K Na:K Ca N Na K Ca N

C 0.021d 2.28c 0.009 4.88a 2.87b 0.077c 1.71b 1.07a 2.85aC + S 0.225a 2.05d 0.109 2.05d 1.89c 0.301a 1.46c 0.84b 1.14dC + S + KN 0.204b 2.76b 0.074 2.96c 2.00c 0.283a 2.02a 0.92b 1.40cC + S + Pro 0.147c 3.31a 0.044 3.92b 4.63a 0.209b 1.59c 0.75c 2.32b

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ncreased all the above-mentioned parameters compared to non-reated stressed plants, but number of stomata was still muchower than that in the control plants. Furthermore, fruit yieldsf plants receiving supplementary proline or KNO3 were stilluch lower than that of control plants (Table 1).Both electrolyte leakage and proline content were increased

n the leaves of melon plants grown at high salinity comparedo the unstressed control plants. But, externally supplied KNO3meliorated this leakage partly and decreased proline content,ut the values were still higher compared to those of controllants. The externally supplied proline reduced the electrolyteeakage largely, but it increased proline content in the leavess expected (Table 2), which could be absorbed and furtheretabolized in plant tissues.The data in Table 3 show that 150 mM NaCl treatment caused

significant increase in Na concentration, but a significanteduction in Ca, N and K concentrations in the leaves and rootsf melon plants. Sodium concentration was diluted by sup-lemented chemicals and by proline application in particular.oncentrations of K, N and Ca were significantly increased byll of the supplied chemicals.

. Discussion

Salinity stress induced lower biomass production, chloro-hyll concentration and fruit yield of melon. This is inccordance with our previous findings in strawberry (Kaya etl., 2001) and cucumber (Kaya et al., 2003). As suggestedy Bernstein (1963), suppression of plant growth under saline

onditions may either be due to osmotic reduction in water avail-bility or to excessive ion (Na+ and Cl−) accumulation in plantissues (Gunes et al., 1995). Toxic ions in the leaves, such asa+ may interfere with phloem loading, and thus translocation

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atments at the P < 0.05 level. C, Control: complete nutrient solution alone; S:in nutrient solution and Pro: 10 mM proline applied foliarly.

f assimilated reserves was inhibited in salinised plants, inde-endent of leaf water potential and relative leaf water contentRWC). Salt treatment induced a reduction in leaf RWC. Theecrease in RWC indicated a loss of turgor that resulted in lim-ted water availability for cell expansion processes (Katerji etl., 1997). The decrease in RWC and chlorophyll content underalinity stress has already been reported (Srivastava et al., 1998)nd the latter attribute has been suggested as one of the impor-ant indicators of salt tolerance in crop plants (Srivastava et al.,998; Raj Kumar Sairam et al., 2002).

High salt (150 mM NaCl) treatment induced significantncreases in electrolyte leakage in the stressed plants comparedith those in the control plants. Similar results were obtainedy Lutts et al. (1996) and Kaya et al. (2002) who reported thatigh salt concentration increased the membrane permeability ofensitive rice varieties and strawberry plants, respectively.

.1. Na/proline interaction

It has been reported that exogenous application of compat-ble solutes offers a valuable tool for studying mechanisms ofalt tolerance. One of these mechanisms depends on the capacityor osmotic adjustment, which allows growth to continue underaline conditions (Heuer, 2003). Salinity treatment (150 mMaCl) resulted in a significant accumulation of proline in melon.roline accumulation in response to stress is widely reported,nd may play a role in stress adaptation within the cell (Gilbertt al., 1998; Ashraf and Foolad, 2007). Evidence for the transportf proline to the root tip, where it accumulates during stress, has

een reported. The rapid accumulation of amino acids duringalinity stress suggests that these compounds may be acting asinks for excess N in relation to the decreased growth occurringuring the imposed stress (Dubey and Pessarakli, 1995). They

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lso play a role in osmotic adjustment, and serve as availableources of carbon and N (Zhang et al., 1997). This availabil-ty could be of special interest at after-stress recovery (Verma,999). Plants may have evolved a mechanism to coordinate syn-hesis, catabolism, and transport activities for the accumulationf proline (Hong et al., 2000). Application of 5 mM prolinemproved the water status of salt stressed melon plants. Suchffects could have been due to inhibition of water efflux viaffects of solutes on membrane stability (Table 2) and reducedranspiration via effects on stomatal regulation (Raghavendrand Reddy, 1987). These findings agree with the results of thexperiment done by Gadallah (1999) in bean plants. Proline alsocts as a major reservoir of energy and N for utilization uponxposure to salinity. Energy for growth and survival may helpn tolerance of salt stress in barley (Girija et al., 2002). In theresent experiment, membrane destabilization caused by salinitytress was greatly reduced in the presence of proline. This soluterobably provides protection against destabilization of proteinsnd membranes (Zhao et al., 1992). Proline-treated plants hadore chlorophyll and produced more biomass, which is in agree-ent with the results of Shaddad (1990) and Gadallah (1995) inaphanus sativus and cotton, respectively.

In the present experiment, exposure of plants to saline nutrientolution significantly increased Na+ concentration in the leavesnd roots of melon plants (Table 3). However, this accumulationas dramatically reduced in both leaves and roots in the presencef proline, suggesting its interference in the process of osmoticdjustment. Consequently, the ratio of Na:K was also signif-cantly different in the presence of 10 mM proline (Table 3).his can be attributed primarily to the ability of roots to exclude

he salt from the xylem sap flowing to the shoot, which wouldmmediately imply better plant growth (Heuer, 2003).

.2. Na/K interaction

It is accepted that competition exists between Na+ and K+

eading to a reduced level of internal K+ at high external NaCloncentration (Botella et al., 1997; Asch et al., 1999; Rus etl., 2004). This can result in high Na:K ratios that reduce plantrowth and eventually become toxic (Schachtman and Lio, 1999;uin et al., 2003). The present study revealed that 150 mM NaCl

nduced a significant decrease in K+ uptake, while the applica-ion of supplied chemicals enhanced K+ uptake by melon plants.t is important to develop an understanding of how plant rootsre capable of distinguishing between essential potassium andotentially toxic sodium ions. Distinguishing between these twoons is not trivial because they are similar in ionic radius andon hydration energies, which are both factors in determiningow these ions move through membrane proteins into cells andhole plants (Hille, 1992). At the whole plant level, the genetic

ontrol of potassium and sodium selectivity has been studied inelation to salinity tolerance. A fundamental role of K uptake,or growth under saline conditions, was demonstrated by the iso-

ation of an Arabidopsis mutant that is hypersensitive to salinitynd deficient in high-affinity K uptake (Wu et al., 1996). Becausef the thermodynamics and also the widely observed interac-ion between Na and K uptake, it is possible that Na enters the

A

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imental Botany 60 (2007) 397–403 401

ell cytoplasm through potassium channels. A large number ofighly selective plasma membrane K channels had been identi-ed in various plant cell types (Amtmann and Sanders, 1999).he possible role of K-selective uptake channels in mediatinga influx has been studied, and most inwardly rectifying chan-els appear to be highly selective for K over Na (Amtmannnd Sanders, 1999). Therefore, it appears unlikely that K-uptakehannels would mediate significant net Na flux into plant cells.

.3. Na/N interaction

In plants in which the primary site of nitrate reduction ishe leaves, photosynthates can be directly utilized for nitrateeduction. Certainly, in such species the cost of N fixation inerms of carbohydrate requirement is high (Marschner, 1995).educed N content of plants grown at high salinity, as observed

n melon in the present study, was reported in a range of crops,uch as cucumber and tomato (Cerda and Martinez, 1988), andarley (Shen et al., 1994). Disruption in the nitrate supply tohe roots under salt stress may lead to a reduced nitrate reduc-ase activity in the leaves and shoot growth rate (Gouia et al.,994; Marschner, 1995). Furthermore, inhibition in N uptakeay occur by NO3

−/Cl− interaction at the sites for ion trans-ort (Cram, 1983), because Na+ results in severe membraneepolarization in plants (Suhayda et al., 1990), which has beeninked to non-competitive inhibition of nitrate uptake (Hawkinsnd Lewis, 1993).

From the results of this experiment, it can be concluded thatroline and KNO3 counteracted the deleterious effects of salinitytress on the parameters investigated, helped the melon plantso avoid Na+ toxicity and improved cell membrane stability anda2+, K+ and N uptake under salinity. Improvement of plantrowth, fruit yield and water status of salt stressed melon plantsakes it possible to recommend the treatment of plants grown

nder saline conditions with these supplied chemicals. Furtherxperiments should be done using combinations of KNO3 androline.

cknowledgements

Authors wish to thank University Mugla and University ofarran (Turkey) for supporting this work. In addition, we would

ike to thank Professor Grant R. Cramer on his suggestions andomments on the draft version of this manuscript.

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