effect of peg-induced drought stress on seed germination of four lentil genotypes

This article was downloaded by: [80.47.125.66]On: 08 July 2014, At: 22:57Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: MortimerHouse, 37-41 Mortimer Street, London W1T 3JH, UK

Journal of Plant InteractionsPublication details, including instructions for authors and subscription information:http://www.tandfonline.com/loi/tjpi20

Effect of PEG-induced drought stress on seedgermination of four lentil genotypesAdele Muscoloa, Maria Sidaria, Umberto Anastasib, Carmelo Santonocetoa & AlbinoMaggioc

a Dipartimento di Agraria, Università “Mediterranea” di Reggio Calabria, Località Feo diVito, 89126 Reggio Calabria, Italyb Dipartimento di Scienze delle Produzioni Agrarie e Alimentari, Università di Catania,Via Valdisavoia 5, 95123 Catania, Italyc Dipartimento di Ingegneria Agraria e Agronomia del Territorio, Università di Napoli“Federico II”, Via Università 100, 80055 Portici, Napoli, ItalyAccepted author version posted online: 20 Aug 2013.Published online: 16 Sep 2013.

To cite this article: Adele Muscolo, Maria Sidari, Umberto Anastasi, Carmelo Santonoceto & Albino Maggio (2014) Effect ofPEG-induced drought stress on seed germination of four lentil genotypes, Journal of Plant Interactions, 9:1, 354-363, DOI:10.1080/17429145.2013.835880

To link to this article: http://dx.doi.org/10.1080/17429145.2013.835880

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) containedin the publications on our platform. Taylor & Francis, our agents, and our licensors make no representationsor warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content.Versions of published Taylor & Francis and Routledge Open articles and Taylor & Francis and RoutledgeOpen Select articles posted to institutional or subject repositories or any other third-party website arewithout warranty from Taylor & Francis of any kind, either expressed or implied, including, but not limited to,warranties of merchantability, fitness for a particular purpose, or non-infringement. Any opinions and viewsexpressed in this article are the opinions and views of the authors, and are not the views of or endorsed byTaylor & Francis. The accuracy of the Content should not be relied upon and should be independently verifiedwith primary sources of information. Taylor & Francis shall not be liable for any losses, actions, claims,proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever causedarising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Terms & Conditions of accessand use can be found at http://www.tandfonline.com/page/terms-and-conditions It is essential that you check the license status of any given Open and Open Select article toconfirm conditions of access and use.

http://www.tandfonline.com/loi/tjpi20

http://www.tandfonline.com/action/showCitFormats?doi=10.1080/17429145.2013.835880

http://dx.doi.org/10.1080/17429145.2013.835880

http://www.tandfonline.com/page/terms-and-conditions

RESEARCH ARTICLE

Effect of PEG-induced drought stress on seed germination of four lentil genotypes

Adele Muscoloa*, Maria Sidaria, Umberto Anastasib, Carmelo Santonocetoa and Albino Maggioc

aDipartimento di Agraria, Universita ‘‘Mediterranea’’ di Reggio Calabria, Localita Feo di Vito, 89126 Reggio Calabria, Italy;bDipartimento di Scienze delle Produzioni Agrarie e Alimentari, Universita di Catania, Via Valdisavoia 5, 95123 Catania, Italy;

cDipartimento di Ingegneria Agraria e Agronomia del Territorio, Universita di Napoli ‘‘Federico II’’, Via Universita100, 80055 Portici, Napoli, Italy

(Received 24 July 2013; accepted 13 August 2013)

Seeds of four lentil genotypes (Castelluccio, Eston, Pantelleria, and Ustica) were subjected to five levels (0, 10, 15,

18, and 21%) of polyethylene glycol (PEG-6000). Germination percentage, root length, tissue water content(WC), a- and b-amylases, a-glucosidase activities, and osmolyte content were evaluated at 24, 48, and 72 h afterstarting the germination test. Water stress reduced seed germination percentage, root length, and seedling WC in

all cultivars to different extent. The increase in proline content and total soluble sugars was greater for Eston andCastelluccio compared to the other genotypes. The activity of the enzymes involved in the germination processdecreased in all cultivars; the activities of a-amylase and a-glucosidase were most negatively affected by osmoticstress, mainly in the drought sensitive Ustica and Pantelleria. Overall, Eston and Castelluccio were able to express

greater drought tolerance and consequently could be used as a valuable resource for breeding programs.

Keywords: amylase activity; drought; lentil; osmotic stress; proline; seed germination

1. Introduction

Worldwide agricultural productivity is subject toincreasing environmental constraints in the form ofabiotic stresses that adversely influence plants growthand development causing crop failure and decreasingaverage yields more than 50% (Buchanan et al. 2000;Bartels & Sunkar 2005; Mittler 2006; Wu et al. 2011).In semiarid environments where lentil is widespread,unfavorable soil moisture at sowing often conditionsseverely seed germination resulting in an irregularseedling emergence, which in turn affects the estab-lishment of a stand, with negative effects on the yield(Mwale et al. 2003; Okcu et al. 2005). For thesereasons, drought tolerance at the germination stagehas specific importance moreover in warm environ-ments most vulnerable to climate change (IPCC2007).

Lentil (Lens culinaris L.), one of the oldestdomesticated plants in the world, originated fromthe near East and central Asia, is traditionallycultivated in the Mediterranean basin (Zohary1972). Seeds of this species are an important sourceof protein for the human diet and the entire biomassof plant is a valued animal feed. Irrigation generallyincreases lentil yield (Salehi et al. 2008), improvingseed size, seed yield, biomass yield, and harvest index(Singh & Saxena 1990; Silim et al. 1993; Khourgamiet al. 2012). Thus, the successful crop establishment insemiarid areas depends on the rapid and uniform seedgermination, which is strictly associated to the abilityof seeds to germinate under low water availability

(Arjenaki et al. 2011). The sequence of events leading

to seed germination and root emergence is governed by

water uptake from the external medium (Kaur et al.

1998; Hodge et al. 2009). Water availability plays a

significant role in enzymatic reactions, solubilisation

and transportation of metabolites, and also as a

reagent in the hydrolytic breakdown of proteins,

lipids, and carbohydrates in the storage tissues of

germinating seeds (Bewley & Black 1994; Biaecka &

Kepczynski 2010). Amylase enzymes play an impor-

tant role during seed germination, hydrolyzing the

endosperm starch into metabolizable sugars, which

provide the energy for the growth of roots and shoots

(Nauriere et al. 1992). The activity of such enzymes is

reduced by water stress with negative effects on

carbohydrate metabolism (Kaur et al. 2000; Zeid &

Shedeed 2006).Selection of plants with a better drought tolerance

is critical in dry environments (Ashraf et al. 1992;

Tuberosa & Salvi 2006). However, controlled and

uniformly repeated simulation of drought in the field

cannot be easily achieved (Shaheen & Hood-Nowotny

2005). The slow progress in developing drought-

resistant cultivars also reflects the lack of a specific

method for screening the large numbers of genotypes

required in breeding for drought (Zeigler & Puckridge

1995). Using natural field conditions is difficult be-

cause rainfall can eliminate water deficits. However, in

vitro drought-screening methods are facilitating pro-

gress in our understanding of drought-resistance traits

and in our selection of drought-resistant genotypes.

*Corresponding author. Email: [email protected]

Journal of Plant Interactions, 2014Vol. 9, No. 1, 354�363, http://dx.doi.org/10.1080/17429145.2013.835880

# 2013 Taylor & Francis

Dow

nloa

ded

by [

80.4

7.12

5.66

] at

22:

57 0

8 Ju

ly 2

014

mailto:[email protected]

http://dx.doi.org/10.1080/17429145.2013.835880

Richards (1978) suggested germination as a useful

criterion in screening for water stress tolerance. Khak-wani et al. (2011) demonstrated that among the sixvarieties of wheat tested, those who were tolerant todrought during in vitro germination tests were simi-

larly tolerant in field conditions. In addition,Agili et al.(2012) confirmed this finding with experiments onsweet potato. Thus, study of the influence of thedrought using osmotic solutions is one of the methods

in the evaluation of resistance during the germinationphase. Exposure to polyethylene glycol (PEG-6000)solutions has been effectively used to mimic drought

stress with limited metabolic interferences as thoseassociated to the use of low molecular weight osmo-lytes that can be taken up by the plant (Hohl &Schopfer 1991). PEG-based in vitro screening for

drought tolerance has been proven to be a suitablemethod to effectively screen large sets of germplasmwith good accuracy (Kulkarni & Deshpande 2007).

Understanding the biochemical mechanisms in-volved in plant drought stress tolerance is still a major

challenge in biology and agriculture to identify atearly stage suitable traits that would support plantbreeders in specific selection programs. The mainobjective of this study was to evaluate the influence of

drought stress on seeds of the four cultivars of lentil,which had previously shown to have diverse level oftolerance to NaCl stress (Sidari et al. 2008), in order

to select the best suitable parents for hybridization inbreeding patterns.

2. Materials and methods

2.1. Plant material, germination conditions, andexperimental design

The following lentil cultivars were studied in thisexperiment. Two salt stress tolerant landraces ‘Pan-

telleria’ and ‘Ustica’: native and cultivated in thehomonymous small islands close to Sicily (SouthernItaly), a local population ‘Castelluccio di Norcia’:

cultivated in Umbria region (Central Italy), and aCanadian commercial variety ‘Eston.’ Seven-month-old seeds (stored at 20918C and 95% R.U.) of eachlentil genotype were used. The seeds were selected for

size homogeneity, surface-sterilized for 20 min in 30%(v/v) H2O2, rinsed and soaked in distilled water for 1h. For each of four genotypes, five replicates of 50-seed were placed on a filter paper in 9-cm Petri dishes

containing 3 cm3 of distilled water or 10, 15, 18, and21% of PEG (MW 6000) concentration correspond-ing to final osmotic potentials of �0.30, �0.51,�0.58, and �0.80 MPa, respectively. We used 10,

15, 18, and 21% of PEG to have an osmotic potentialcomparable to that of NaCl at the concentrations of50, 100, 150, and 200 mM that we tested on seed

germination of the same cultivars in a previous work(Sidari et al. 2008), in order to evaluate similarity ordifferences in the metabolic traits of salt and droughtresistance. The Petri dishes were sealed with Parafilm

to prevent evaporation and kept accordingly to acompletely randomized design in a growth chamber ata temperature of 25918C in the dark with a relativehumidity of 70%. Seeds were considered germinatedwhen the radicle had extended for at least 2 mm. Thewater content (WC) was measured and expressed as apercentage according to the formula WC (%)�(Fresh Weight � Dry Weight/Fresh Weight)�100.Root length (cm) was also measured and for each offour genotypes, five replicates were used.

2.2. Enzyme activity

The activities of a-amylase, b-amylase, and a-gluco-sidase were determined in the crude extracts of eachcultivar. The seeds of each cultivar and for each PEGtreatment (0, 10, 15, 18, and 21%) were homogenizedin a chilled mortar with distilled water 1:4 (w/v) andcentrifuged at 14,000 g for 30 min. The supernatantswere filtered through a single layer of muslin clothand were used for a-amylase (EC 3.2.1.1) (Steup1988), b-amylase (EC 3.2.1.2) (Steup 1988), anda-glucosidase (EC 3.2.1.20) (Bergmeyer et al. 1983)activity determination.

For a-amylase, a mixture of 3 ml soluble starch(2% v/v) and 3 ml extract was incubated for 60 min at308C. After incubation, an equal volume of alkalinecolor reagent was added to 1-ml incubation mixture,mixed and heated for five min in a boiling water bath.The absorbance at 546 nm was measured against ablank (1 ml H2O plus 1 ml alkaline reagent). Thestandard curve was obtained by using different con-centrations of maltose in the range of 0�1.5 mmol l�1.The alkaline color reagent was prepared by dissolving1 g of 3,5-dinitrosalycylic acid in a mixture of 40 ml1 N NaOH solution and 30 ml H2O. Solid potassiumsodium tartrate was added and dissolved. The mixturewas brought to a final volume of 100 ml (Steup 1988).b-amylase was determined as described above butsoluble starch was replaced by amylopectin. Fora-glucosidase detection, the assay buffer consisted of50 mM Na-acetate, pH 5.2, containing 10 mM CaCl2.The substrate was 10 mmol l�1 maltose. The sampleswere incubated for 60 min. The release of glucose wasfollowed by measuring the changes in NADPH at340 nm in a coupled enzyme reaction of hexokinaseand glucose-6-P dehydrogenase. For each treatment,five replicates were used.

2.3. Osmolyte content

To detect free proline content samples (0.3 g) includ-ing 5 ml of 3% sulfosalicylic acid were homogenizedand centrifuged at 3000 rpm for 20 min. The super-natant was added to 2 ml of glacial acetic acid with2 ml acidic ninhydrin. The mixture was heated at1008C for 25 min. After the liquid was cooled, themixture was added to 4 ml toluene. The absorbance ofthe extracts was read at 520 nm (Bates et al. 1973). Thetotal soluble sugars were determined with the

Journal of Plant Interactions 355

Dow

nloa

ded

by [

80.4

7.12

5.66

] at

22:

57 0

8 Ju

ly 2

014

anthrone method (Yemn & Willis 1954). For eachtreatment, five replicates were used.

2.4. Measurement times and statistical analysis

Root length, water content (WC), enzyme activity,and osmolyte content were measured at 24, 48, and72 h after the start of the test. The data werestatistically analyzed separately for each time by atwo-way ANOVA according to the adopted experi-mental design combining PEG concentrations andgenotypes. The germination percentage data werepreviously subjected to arcsine transformation andwere reported in tables as untransformed values. Thedifferences between the means were compared by theleast significant difference (LSD) test (p50.05).

3. Results

Germination was significantly affected by the osmoticpotential, by cultivars and their interaction (Table 1).Germination of all cultivars started 24 h after sowing.The final germination percentage of the control (0%PEG) reached 100% for each cultivar but withdifferent time. An increase in PEG stress markedlydecreased the germination percentage of all cultivarscompared to their relative controls. Seventy-twohours after sowing, the germination percentage ofCastelluccio and Eston at the highest PEG concen-trations (18% and 21%) was higher than that ofPantelleria and Ustica.

The effects of drought stress, cultivars, and theirinteraction were also significant on root length. Root

length decreased, increasing water stress and time butwith different extent (Table 2). By increasing PEGconcentrations a different behavior among the culti-vars was observed. Eston and Castelluccio showeda greater radicle elongation especially at 72 h incomparison to Pantelleria and Ustica. The greatestradicle reduction was observed in Ustica and Pantel-leria at 48 and 72 h in presence of PEG at theconcentrations of 18 and 21%.

In all cultivars, in the absence of stress, the WCincreased over time (Table 3). The increase in bothduration and intensity of osmotic stress caused agradual decrease in the WC in each cultivar comparedto controls. The presence of PEG at different con-centrations differently affected the cultivars over time.Increasing the duration of stress and the time, the WCdecreased in Pantelleria and Ustica and increased inEston and Castelluccio. The lowest WCs were de-tected at 72 h in Ustica and Pantelleria in presence ofPEG at the highest (18% and 21%) concentrations.

Total soluble sugar content decreased during theexperimental time in Pantelleria and Ustica seeds. Onthe contrary, as a consequence of the increasingdrought stress and time, in Eston and Castelluccio, agradual increase in total soluble sugars was observed(Figure 1A)

Significant increase in free proline content wasalso observed in seeds of all genotypes under waterstress. Eston and Castelluccio under drought condi-tions, accumulated more proline than Pantelleria andUstica (Figure 1B).The highest amount of proline wasdetected in the above two cultivars in presence ofPEG at the concentrations of 18 and 21% at 72 h.

Table 1. Germination (%) of lentil seeds as affected by genotype (G), PEG concentration (C) factors and their interaction

(G�C) after 24, 48, and 72 h.

Time (h)Factors PEG concentration (C)

Genotype (G) 0 10 15 18 21 (%)

24 LSD (G�C) 3.4 Mean (C)

Eston 97 72 33 11 8 44bCastelluccio 98 47 15 4 3 33cUstica 100 72 47 35 5 52a

Pantelleria 100 71 44 35 1 51aMean (G) 99a 66b 35c 21d 4e

48 LSD (G�C) 3.5 Mean (C)Eston 99 80 71 67 44 72a

Castelluccio 98 48 35 30 18 46cUstica 100 76 53 49 27 61bPantelleria 100 76 57 42 28 60bMean (G) 99a 70b 54c 47d 29e

72 LSD (G�C) 2.3 Mean (C)Eston 100 100 93 91 76 92aCastelluccio 100 71 64 61 47 69bUstica 100 81 61 51 25 64c

Pantelleria 100 81 64 52 28 65cMean (G) 100a 84b 71c 64d 44e

Note: At each time (h), means followed by the same letter, within each factor, are not significantly different according to LSD (p50.05).

Means of interaction (G�C) were compared according to LSD (p50.05).

356 A. Muscolo et al.

Dow

nloa

ded

by [

80.4

7.12

5.66

] at

22:

57 0

8 Ju

ly 2

014

The values of a-amylase, b-amylase, and

a-glucosidase activities were constitutively different

in the seeds of the four cultivars (Tables 4�6).Theactivity of these enzymes decreased in a dose depen-

dent manner, differing among the cultivars. The

activities of a-, b-amylase, and a-glucosidase in

stressed seeds of Eston and Castelluccio were higher,

compared to Ustica and Pantelleria with respect to

time and stress level. The activities of these enzymes

showed a greater decreasing trend in Pantelleria and

Ustica in presence of PEG at the concentrations of

18% and 21%, already 24 h after sowing. Among

the enzymes involved in the germination process,

a-amylase and a-glucosidase were the most negatively

Table 2. Root length (cm) of lentil seeds as affected by genotype (G), PEG concentration (C) factors and their interaction

(G�C) after 24, 48, and 72 h.


Genotype (G) 0 10 15 18 21 (%)

24 LSD (G�C) 0.31 Mean (C)

Eston 0.69 0.36 0.23 0.17 0.12 0.32aCastelluccio 0.40 0.24 0.14 0.07 0.02 0.17dUstica 0.52 0.27 0.20 0.15 0.08 0.25c

Pantelleria 0.69 0.39 0.24 0.11 0.09 0.30bMean (G) 0.57a 0.31b 0.20c 0.12d 0.08e

48 LSD (G�C) 0.06 Mean (C)Eston 2.51 1.34 1.00 0.68 0.24 1.15a

Castelluccio 1.10 0.83 0.76 0.52 0.20 0.68dUstica 2.03 0.86 0.65 0.34 0.18 0.81cPantelleria 2.85 0.92 0.77 0.47 0.18 1.04bMean (G) 2.12a 0.99b 0.80c 0.50d 0.20e

72 LSD (G�C) 0.06 Mean (C)Eston 4.31 2.22 1.74 1.02 0.83 2.02aCastelluccio 3.94 1.60 1.35 0.74 0.53 1.63bUstica 4.15 1.49 1.12 0.42 0.22 1.48d

Pantelleria 4.95 1.53 0.87 0.33 0.21 1.58cMean (G) 4.33a 1.71b 1.27c 0.63d 0.44e


Means of interaction (G�C) were compared according to LSD (p50.05).

Table 3. WC (%) of lentil seeds as affected by genotype (G), PEG concentration (C) factors and their interaction (G�C)after 24, 48, and 72 h.


Genotype (G) 0 10 15 18 21 (%)

24 LSD p50.05 (G�C) 2.3 Mean (C)Eston 19 16 17 14 14 16c

Castelluccio 19 16 17 15 15 16cUstica 25 24 21 19 15 21bPantelleria 38 22 21 22 21 25a

Mean (G) 25a 20b 19b 17c 16c

48 LSD p50.05 (G�C) 2.5 Mean (C)Eston 39 27 28 27 21 29aCastelluccio 31 28 27 29 21 27bUstica 33 34 24 20 16 25c

Pantelleria 37 37 19 19 17 26cMean (G) 35a 32b 25c 24c 19d

72 LSD p50.05 (G�C) 2.9 Mean (C)Eston 40 34 30 26 24 31a

Castelluccio 42 26 25 25 22 28bUstica 35 31 14 12 10 20cPantelleria 45 35 13 7 7 21c

Mean (G) 40a 32b 20c 17d 16e


Means of interaction (G�C) are compared according to LSD (p50.05).


Dow

nloa

ded

by [

80.4

7.12

5.66

] at

22:

57 0

8 Ju

ly 2

014

affected by drought stress especially in Ustica and

Pantelleria cultivars.

4. Discussion

Drought is a multifaceted stress condition that causes

serious crops yield limitations depending on plant

growth stage, stress duration, and severity. Germina-tion is the most critical and sensitive stage in the lifecycles of plants (Ahmad et al. 2009) and the seedsexposed to unfavorable environmental conditionssuch as drought may compromise the subsequentseedling establishment (Albuquerque & Carvalho2003; Soleymani et al. 2012). Genetic variability

Tot

al s

olub

re c

arbo

hydr

ates

[m

g g–

1 (d

w)]

0

20

40

60

80

PantelleriaUsticaEstonCastelluccio

0 h

Tot

al s

olub

le c

arbo

hydr

ates

[m

g g–

1 (d

w)]

0

20

40

60

80

24 h

Tot

al s

olub

le c

arbo

hydr

ates

[m

g g–

1 (d

w)]

0

20

40

60

80

48 h

PEG concentration %

0 5 10 15 20

Tot

al s

olub

le c

arbo

hydr

ates

[m

g g–

1 (d

w)]

0

20

40

60

80

72 h

Prol

ine

[µg

g–1

(dw

)]

0

200

400

6000 h

Prol

ine

[µg

g–1

(dw

)]

0

200

400

600 24 h

Prol

ine

[µg

g–1

(dw

)]

0

200

400

60048 h

PEG concentration %

0 5 10 15 20

Prol

ine

[µg

g–1

(dw

)]

0

200

400

60072 h

A B

Pantelleria

Ustica

Eston

Castelluccio

Figure 1. Total soluble carbohydrates (A) and proline content (B) in Pantelleria, Ustica, Eston, and Castelluccio lentilgenotypes seeds in 0, 10, 15, 18, and 21% PEG 6000 at 0, 24, 48, and 72 h. Vertical bars indicate SD (n�5).


Dow

nloa

ded

by [

80.4

7.12

5.66

] at

22:

57 0

8 Ju

ly 2

014

within a species offers a valuable tool for studying

mechanism of drought tolerance. Our results high-

lighted significant differences among the cultivars

exposed to drought stress with a remarkably de-

creased and delayed germination in Ustica and

Pantelleria. These results are consistent with those

of other studies that have reported that high con-

centrations of PEG reduce the final germination

percentages of lentil (Siahsar et al. 2010; Jamaati-e-

Somarin & Zabihi-e-Mahmoodabad 2011). In Med-

iterranean semiarid regions, the topsoil WC during

the dry season is drastically reduced, sometimes to

less than 1%, which is very close to the permanent

wilting point estimated for xerophytes (Larcher 1995;

Table 4. a-amylase activity (mmoles of reducing sugars formed min�1 g�1 f.w.) of lentil seeds as affected by genotype (G),

PEG concentration (C) factors and their interaction (G�C) after 24, 48, and 72 h.


Genotype (G) 0 10 15 18 21 (%)

24 LSD p50.05 (G�C) 0.49 Mean (C)

Eston 16.12 17.42 17.04 15.49 12.13 15.64cCastelluccio 17.18 16.77 12.41 11.90 11.12 13.88dUstica 26.10 18.97 19.07 14.47 7.70 17.26b

Pantelleria 36.42 31.35 25.28 17.01 7.39 23.49aMean (G) 24.00a 21.12b 18.45c 14.71d 9.58e

48 LSD p50.05 (G�C) 0.21 Mean (C)Eston 16.41 15.31 14.09 13.22 11.13 14.03c

Castelluccio 15.52 14.06 13.37 12.06 11.88 13.37dUstica 27.06 20.09 15.10 10.47 5.69 15.68bPantelleria 36.05 21.07 13.91 11.22 6.64 17.78aMean (G) 23.76a 17.63b 14.11c 11.74d 8.84e

72 LSD p50.05 (G�C) 0.48 Mean (C)Eston 16.42 16.74 15.50 14.70 12.73 15.22cCastelluccio 12.11 11.97 11.52 11.46 11.06 11.62dUstica 24.93 22.47 16.40 11.47 7.70 16.59b

Pantelleria 36.14 25.20 21.12 18.60 6.73 21.56aMean (G) 22.40a 19.09b 16.13c 14.06d 9.55e



Table 5. b-amylase activity (mmoles of reducing sugars formed min�1 g�1 f.w.) of lentil seeds as affected by genotype (G),

PEG concentration (C) factors and their interaction (G�C).


Genotype (G) 0 10 15 18 21 (%)

24 LSD p50.05 (G�C) 0.09 Mean (C)

Eston 0.75 0.65 0.54 0.43 0.38 0.55aCastelluccio 0.69 0.62 0.51 0.44 0.36 0.53aUstica 1.03 0.55 0.45 0.22 0.19 0.49b

Pantelleria 1.06 0.48 0.41 0.24 0.15 0.47bMean (G) 0.88a 0.58b 0.48c 0.33d 0.27e

48 LSD p50.05 (G�C) 0.33 Mean (C)Eston 0.91 0.88 0.84 0.80 0.64 0.81a

Castelluccio 0.80 0.71 0.65 0.60 0.56 0.67bUstica 1.40 0.75 0.45 0.34 0.23 0.63cPantelleria 1.37 0.73 0.43 0.32 0.22 0.61cMean (G) 1.12a 0.77b 0.60c 0.51d 0.41e

72 LSD p50.05 (G�C) 0.13 Mean (C)Eston 1.17 1.09 0.80 0.73 0.75 0.91aCastelluccio 1.02 0.86 0.72 0.62 0.55 0.75cUstica 1.55 1.35 0.60 0.45 0.24 0.83b





Dow

nloa

ded

by [

80.4

7.12

5.66

] at

22:

57 0

8 Ju

ly 2

014

Padilla & Pugnaire 2007; Munodawafa 2012). Con-

sequently, plant traits related to water uptake are of

paramount importance for explaining plant persis-

tence in Mediterranean-type ecosystems (Valladares

et al. 2004). The ability to develop extensive root

systems contributes to differences among cultivars for

drought tolerance and root length is considered an

important trait in selection of drought resistant

cultivars (Turner 1997; Abd Allah et al. 2010).

Thus, root morphology and/or growth rate may be

instrumental to select drought tolerant varieties

(Wahbi & Gregory 1995; Malik et al. 2002). In our

study, total root length decreased with increasing

PEG concentrations more in Pantelleria and Ustica

than in the other two genotypes, indicating a higher

sensitivity to osmotic stress in these two landraces.

The root length reduction in Pantelleria and Ustica

under drought stress may be associated to a reduced

cellular division and elongation during germination

(Frazer et al. 1990). This hypothesis is supported

by the results of enzymatic activities involved in the

germination process and by the results of WC.

Water availability is usually the limiting factor for

the germination of non-dormant seeds, affecting

the percentage, speed, and uniformity of emergence

(Marcos-Filho 2005; Kaydan & Yagmur 2008). A

threshold level of hydration is required for the

synthesis of hydrolytic enzymes which are responsible

for the hydrolysis of stored substrates. The hydro-

lyzed products are utilized in seedling tissue synthesis

and radicle elongation (Canas et al. 2006). In lentil

seeds, the role of providing utilizable substrates is

taken over mainly by amylases. Inhibition of seed

germination (Haouari et al. 2013) is directly related to

reserve mobilization, energy production through

respiration, enzyme and hormonal activity, and

dilution of the protoplasm to increase metabolism

for successful embryonic growth (McDonald 2007).

The PEG treatment decreased WC in the seeds of the

studied lentil genotypes, as well documented also for

other species under similar experimental conditions

(Bajji et al. 2000; Guoxiong et al. 2002; Wu et al.

2011; Haouari et al. 2013). Seeds of Castelluccio and

Eston had higher WC compared to those of Ustica

and Pantelleria indicating that the former genotypes

have a more efficient water uptake/control system

that can be related to the increase in the free proline

content and total soluble carbohydrates, suggesting

that drought tolerance ability of these two last

landraces appears to be associated to the accumula-

tion of osmolytes which improved their water status.

The osmolyte content increase is one of the self-

defense reactions during water stress in seeds and

plants protecting the enzyme system. Drought and

salt stress have been reported to limit the mobilization

of starchy endosperm reserves in several species, as a

result of inhibition of different enzymatic activities

(Ashraf & Foolad 2005; Besma & Mounir 2010;

Biaecka & Kepczynski 2010). Starch mobilization

results from simultaneous activities of a-amylase,

b-amylase, and a-glucosidase. In germinating seeds,

starch degradation is initiated by a-amylase (Kaur

et al. 2005), that produces soluble oligosaccharides

from starch and these are then hydrolyzed by

b-amylase to liberate maltose. Finally, a-glucosidasebreaks down maltose into glucose, the main respira-

Table 6. a-glucosidase activity (mmoles of reducing sugars formed min�1 g�1 f.w.) of lentil seeds as affected by genotype (G),

PEG concentration (C) factors and their interaction (G�C) after 24, 48, and 72 h.


Genotype (G) 0 10 15 18 21 (%)

24 LSD p50.05 (G�C) 0.003 Mean (C)

Eston 0.270 0.284 0.297 0.212 0.185 0.250aCastelluccio 0.220 0.187 0.150 0.130 0.122 0.196bUstica 0.303 0.292 0.192 0.062 0.060 0.182c

Pantelleria 0.309 0.265 0.143 0.059 0.055 0.166dMean (G) 0.273a 0.257b 0.195c 0.115d 0.105e

48 LSD p50.05 (G�C) 0.009 Mean (C)Eston 0.282 0.296 0.197 0.166 0.140 0.216a

Castelluccio 0.263 0.191 0.148 0.142 0.125 0.173bUstica 0.321 0.234 0.153 0.084 0.024 0.163cPantelleria 0.310 0.240 0.145 0.065 0.025 0.157cMean (G) 0.294a 0.240b 0.160c 0.114d 0.078e

72 LSD p50.05 (G�C) 0.012 Mean (C)Eston 0.299 0.305 0.276 0.208 0.189 0.255aCastelluccio 0.256 0.247 0.215 0.198 0.186 0.220bUstica 0.338 0.207 0.061 0.041 0.011 0.131c





Dow

nloa

ded

by [

80.4

7.12

5.66

] at

22:

57 0

8 Ju

ly 2

014

tory substrate (Sticklen 2008), with release of theenergy required for essential metabolic functions(Nauriere et al. 1992). Consistently, the activity ofa- and b-amylases in germinating seeds is reduced bywater stress (Zeid & Shedeed 2006). Our resultsconfirmed that the amylase activities in lentil seedsdecreased under PEG induced drought stress, andsuggested that the variation in stress sensitivity ofcontrasting lentil genotypes may be linked to theirability to osmoregulate under stress, which causea strong decrease in WC affecting the hydro-lytic enzyme activities, particularly a-amylase anda-glucosidase levels highlighting the greatest decreasein the most drought sensitive Ustica and Pantelleriaseeds.

The germination of Ustica and Pantelleria, whichare previously identified as NaCl resistant genotypes(Sidari et al. 2007, 2008), was lower than Eston andCastelluccio at the same iso-osmotic PEG concentra-tions. These results indicate that mechanisms mediat-ing drought stress tolerance at germination stage aredifferent from those that mediate salt stress tolerance(Munns 2002). Since PEG does not enter to seeds(Khajeh-Hosseini et al. 2003; Mehra et al. 2003),these differences can be specifically associated tomechanisms that control ion homeostasis and toxicity(Bohnert et al. 1999). Identifying drought resistantcultivars of lentil at early growth stages is essential tocultivate this crop in arid and/or semiarid environ-ments where the survival of other species would bedifficult.

Taken together, these findings suggest that seedgermination, WC, and root length can be used astraits for rapid selection of drought tolerant cultivars.Eston and Castelluccio can be considered as valuabledrought tolerant germplasm. Our data highlighted anopposite response to salt and drought tolerancebecause the two genotypes resulting resistant todrought (Eston and Castelluccio) were found saltsensitive (Sidari et al. 2008) whereas the two drought-sensitive genotypes (Ustica and Pantelleria) werefound NaCl tolerant by the same authors. We canconclude that all these lentil genotypes could be usednot only in breeding programs to improve toleranceto both drought and salinity stress with the aim toincrease the probability of successful legume estab-lishments in arid or semiarid environments but also tobe cultivated in environments where water scarcity isa frequent constraint.

Acknowledgments

This research was supported by ‘‘Mediterranea’’ University

of Reggio Calabria-Italy, Programmi di Ricerca Scientifica

RDB-2011.

References

Abd Allah AA, Badawy Shimaa A, Zayed BA, ElGohary

AA. 2010. The role of root system traits in the drought

tolerance of rice (Oryza sativa L.). World Acad Sci Eng

Technol. 68:1378�1382.Agili S, Nyende B, Ngamau K, Masinde P. 2012. Selection,

yield evaluation, drought tolerance indices of orange-

flesh sweet potato (Ipomoea batatas Lam) Hybrid

Clone. J Nutr Food Sci. 2:138.Ahmad S, Ahmad R, Ashraf MY, Ashraf M, Waraich EA.

2009. Sunflower (Helianthus annuus L.) response to

drought stress at germination and growth stages. Pak J

Bot. 41:647�654.Albuquerque FMCD, Carvalho NMD. 2003. Effect of type

of environmental stress on the emergence of sunflower

(Helianthus annuus L.), soyabean (Glycine max (L.)

Merril) and maize (Zea mays L.) seeds with different

levels of vigor. Seed Sci Technol. 31:65�467.Arjenaki FG, Dehaghi MA, Jabbari R. 2011. Effects of

priming on seed germination of Marigold (Calendula

officinalis). Adv Environ Biol. 5:276�280.Ashraf M, Bokhari H, Cristiti SN. 1992. Variation in

osmotic adjustment of lentil (Lens culinaris, Medik) in

response to drought. Acta Bot Neerl. 41:51�62.Ashraf M, Foolad MR. 2005. Pre-sowing seed treatment �

a shotgun approach to improve germination growth

and crop yield under saline and non-saline conditions.

Advan Agron. 88:223�271.Bajji M, Lutts S, Kinet JM. 2000. Physiological changes after

exposure to and recovery from polyethylene glycol-

induced water deficit in roots and leaves of durum

wheat (Triticum durum Desf.) cultivars differing in

drought resistance. J Plant Physiol. 157:100�108.Bartels D, Sunkar R. 2005. Drought and salt tolerance in

plants. Critical Rev Plant Sci. 24:23�58.Bates LS, Waldren RP, Teare ID. 1973. Rapid determina-

tion of free proline for water stress studies. Plant Soil.

39:205�207.Bergmeyer HU, Grabl M, Walter HE. 1983. Enzymes. In:

Bergmeyer HU, Bergmeyer J, Grabl M, editors.

Methods of enzymatic analysis. Weinheim: Verlag

Chemie; p. 126�328.Besma BD, Mounir D. 2010. Salt stress induced changes in

germination, sugars, starch and enzyme of carbohy-

drate metabolism in Abelmoschus esculentus L.

(Moench.) seeds. Afr J Agric Res. 5:1412�1418.Bewley JD, Black M. 1994. Seeds: physiology of develop-

ment and germination. New York: Plenum Press.Biaecka B, Kepczynski J. 2010. Germination, a-, b-amylase

and total dehydrogenase activities of amaranthus

caudatus seeds under water stress in the presence of

ethephon or gibberellin A3. Acta Biol Cracov Ser Bot.

52:7�12.Bohnert HJ, Su H, Shen B. 1999. Molecular mechanisms

of salinity tolerance. In: Shinozaki K, Yamaguchi-

Shinozaki K, editors. Molecular responses to cold,

drought, heat and salt stress in higher plants. Austin,

TX: Landes Company; p. 29�60.Buchanan BB, Gruissem W, Jones RL. 2000. Biochemistry

and molecular biology of plants. Rockville, MD:

American Society of Plant Physiologists.Canas RA, Canovas FM, Canton FR. 2006. High levels of

asparagines synthetase in hypocotyls of pine seedlings

suggest a role of the enzyme in re-allocation of seed

scord nitrogen. Planta. 224:83�95.Frazer TE, Silk WK, Rost TL. 1990. Effect of low water

potential on cortical cell length in growing region of

maize roots. Plant Physiol. 93:648�651.


Dow

nloa

ded

by [

80.4

7.12

5.66

] at

22:

57 0

8 Ju

ly 2

014

Guoxiong C, Krugman T, Fahima T, Korol AB, Nevo E.

2002. Comparative study on morphological and phy-

siological traits related to drought resistance between

xeric and mesic Hordeum spontaneum lines in Israel.

Barley Gen Newsl. 32:22�33.Haouari CC, Nasraoui AH, Carrayol E, Gouia H. 2013.

Variations in a-, b-amylase and a-glycosidase activitiesin two genotypes of wheat under NaCl salinity stress.

Afr J Agricult Res. 8:2038�2043.Hodge A, Berta G, Doussan C, Merchan F, Crespi M.

2009. Plant root growth, architecture and function.

Plant Soil. 321:153�187.Hohl M, Schopfer P. 1991. Water relations of growing

maize coleoptiles. Comparison between mannitol and

polyethylene glycol 6000 as external osmotica for

adjusting turgor pressure. Plant Physiol. 95:716�722.IPCC. 2007. Climate change 2007: synthesis report, con-

tribution of working groups I, II and III to the fourth

assessment report of the intergovernmental panel on

climate change. In: Pachauri RK, Reisinger A, editors.

Core writing team. Geneva: IPCC; 104 p.Jamaati-e-Somarin S, Zabihi-e-Mahmoodabad R. 2011.

Evaluation of drought tolerance indices of lentil

varieties. Adv Environ Biol. 5:581�584.Kaur S, Gupta AK, Kaur N. 1998. Gibberellic acid and

kinetin partially reverse the effect of water stress on

germination and seedling growth. Plant Growth Regul.

25:29�33.Kaur S, Gupta AK, Kaur N. 2000. Effect of GA3, kinetin

and indole acetic acid on carbohydrate metabolism in

chickpea seedlings germinating under water stress.

Plant Growth Regul. 30:61�70.Kaur R, Liu X, Goerup O, Zhang A, Yuan X, Balk SP,

Schneider MC, Lu ML. 2005. Activation of p 21-

activated kinase 6 by MAP kinase 6 and p 38 MAP

kinase. J Bio Chem. 280:3323�3330.Kaydan D, Yagmur M. 2008. Germination, seedling

growth and relative water content of shoot in different

seed sizes of triticale under osmotic stress of water and

NaCl. Afr J Biotechnol. 7:2862�2868.Khajeh-Hosseini M, Powell AA, Bingham IJ. 2003. The

interaction between salinity stress and seed vigour

during germination of soybean seeds. Seed Sci Tech-

nol. 31:715�725.Khakwani AA, Dennett MD, Munir M. 2011. Drought

tolerance screening of wheat varieties by inducing

water stress conditions. Songklanakarin J Sci Technol.

33:135�142.Khourgami A, Maghooli E, Rafiee M, Bitarafan Z. 2012.

Lentil response to supplementary irrigation and plant

density under dry farming condition. Int J Sci Adv

Technol. 2:51�55.Kulkarni M, Deshpande U. 2007. In Vitro screening of

tomato genotypes for drought resistance using poly-

ethylene glycol. Afr J Biotechnol. 6:691�696.Larcher W. 1995. Physiological plant ecology. Berlin:

Springer.Malik AI, Colmer TD, Lambers H, Setter TL, Schorte-

meyer M. 2002. Short-term waterlogging has long-term

effects on the growth and physiology of wheat. New

Phytol. 153:225�236.Marcos-Filho JM. 2005. Fisiologia de sementes de plantas

cultivadas [Seed physiology of cultivated 480 plants].

Piracicaba: FEALQ.

McDonald MB. 2007. Seed moisture and the equilibrium

seed moisture content curve. J Seed Technol. 29:7�18.Mehra V, Tripathi J, Powell AA. 2003. Aerated hydration

treatment improves the response of Brassica juncea and

Brassica campestris seeds to stress during germination.

Seed Sci Technol. 31:57�70.Mittler R. 2006. Abiotic stress, the field environment and

stress combination. Trends Plant Sci. 11:15�19.Munodawafa A. 2012. The effect of rainfall characteristics

and tillage on sheet erosion and maize grain yield in

semiarid conditions and granitic sandy soils of Zim-

babwe. Appl Environ Soil Sci. 2012:1�8.Munns R. 2002. Comparative physiology of salt and water

stress. Plant Cell Environ. 25:239�250.MwaleS,HamusimbiC,MwansaK.2003.Germination, emer-

gence and growth of sunflower (Helianthus annuus L.) in

response to osmotic seed priming. Seed Sci Technol.

31:199�206.Nauriere C, Doyen C, Thevenot C, Dauffant J. 1992.

b-amylase in cereals: study of the maize b-amylase

system. Plant Physiol. 100:887�893.Okcu G, Kaya MD, Atak M. 2005. Effects of salt and

drought stresses on germination and seedling growth

of pea (Pisum sativum L.). Turk J Agric For. 29:237�242.

Padilla FM, Pugnaire FI. 2007. Rooting depth and soil

moisture control Mediterranean woody seedling survi-

val during drought. Funct Ecol. 21:489�495.Richards RA. 1978. Variation between and within species

of rapeseed (Brassica campestris and B. napus) in

response to drought stress. III. Physiological and

physicochemical characters. Aust J Agric Res. 29:

491�501.Salehi M, Haghnazari A, Shekari F, Faramarzi A. 2008.

The study of seed yield and seed yield components of

lentil (Lens culinarisMedik) under normal and drought

stress conditions. Pak J Biol Sci. 11:758�62.Shaheen R, Hood-Nowotny RC. 2005. Effect of drought

and salinity on carbon isotope discrimination in wheat

cultivars. Plant Sci. 168:901�909.Siahsar BA, Ganjali S, Allahdoo M. 2010. Evaluation of

drought tolerance indices and their relationship with

grain yield of lentil lines in drought-stressed and

irrigated environments. Aust J Basic Appl Sci.

4:4336�4346.Sidari M, Muscolo A, Anastasi U, Preiti G, Santonoceto C.

2007. Response of four genotypes of lentil to salt stress

conditions. Seed Sci Technol. 35:497�503.Sidari M, Santonoceto C, Anastasi U, Preiti G, Muscolo A.

2008. Variations in four genotypes of lentil under

NaCl-salinity stress. Am J Agric Biol Sci. 3:410�416.Silim SN, Saxena MC, Erskine W. 1993. Adaptation of

lentil to the Mediterranean environment. II. Response

to moisture supply. Exp Agric. 29:21�28.Singh KB, Saxena MC. 1990. Studies on drought tolerance:

Annual report. Aleppo, Syria: ICARDA.Soleymani A, Hesam M, Shahrajabian H. 2012. Study of

cold stress on the germination and seedling stage and

determination of recovery in rice varieties. Int J Biol.

4:23�30.Steup M. 1988. Starch degradation. In: Preiss J, editor. The

biochemistry of plants: a comprehensive treatise. San

Diego, CA: Academic Press; p. 255�296.


Dow

nloa

ded

by [

80.4

7.12

5.66

] at

22:

57 0

8 Ju

ly 2

014

Sticklen MB. 2008. Plant genetic engineering for biofuelproduction: towards affordable cellulosic ethanol. NatRev Genet. 9:433�443.

Tuberosa R, Salvi S. 2006. Genomics-based approaches toimprove drought tolerance of crops. Trends Plant Sci.11:405�412.

Turner NC. 1997. Further progress in crop water relations.Adv Agron. 58:293�338.

Valladares F, Vilagrosa A, Penuelas J, Ogaya R, Camarero

JJ, Corchera L, Siso S, Gil-Pelegrın E. 2004. Estreshıdrico: ecofisiologıa y escalas de la sequıa [Waterstress: ecophysiology and scales of drought]. In:Valladeres F, editor. Ecologıa del BosqueMediterraneo

en un Mundo Cambiante. Madrid: EGRAF, SA;p. 163�190.

Wahbi A, Gregory PJ. 1995. Growth and development of

young roots of barley (Hordeum vulgare L.) genotypes.Ann Bot. 75:533�539.

Wu C, Wang Q, Xie B, Wang Z, Cui J, Hu T. 2011. Effects

of drought and salt stress on seed germination of three

leguminous species. Afr J Biotechnol. 10:17954�17961.Yemn EW, Willis AJ. 1954. The estimation of carbohy-

drates in plant extracts by anthrone. Biochem J.

57:508�514.Zeid IM, Shedeed ZA. 2006. Response of alfalfa to

putrescine treatment under drought stress. Biol Plant.

50:635�640.Zeigler RS, Puckridge DW. 1995. Improving sustainable

productivity in rice based rainfed lowland systems of

South and Southeast Asia. Feeding four billion people:

the challenge for rice research in the 21st century.

GeoJournal. 35:307�324.Zohary D. 1972. The wild progenitor and place of origin

of the cultivated lentil Lens culinaris. Econ Bot.

26:326�332.


Dow

nloa

ded

by [

80.4

7.12

5.66

] at

22:

57 0

8 Ju

ly 2

014

effect of peg-induced drought stress on seed germination of four lentil genotypes

Documents