REGULAR ARTICLE

Evaluation of physiological traits for improving droughttolerance in faba bean (Vicia faba L.)

Habib ur Rahman Khan Æ W. Link ÆT. J. Hocking Æ F. L. Stoddard

Received: 25 September 2006 / Accepted: 6 February 2007 / Published online: 3 March 2007� Springer Science+Business Media B.V. 2007

Abstract Among grain legumes, faba bean is

becoming increasingly popular in European agri-

culture due to recent economic and environmen-

tal interests. Faba bean can be a highly productive

crop, but it is sensitive to drought stress and yields

can vary considerably from season to season.

Understanding the physiological basis of drought

tolerance would indicate traits that can be used as

indirect selection criteria for the development of

cultivars adapted to drought conditions. To assess

genotypic variation in physiological traits associ-

ated with drought tolerance in faba bean and to

determine relationships among these attributes,

two pot experiments were established in a growth

chamber using genetic materials that had previ-

ously been screened for drought response in the

field. Nine inbred lines of diverse genetic back-

grounds were tested under adequate water supply

and limited water conditions. The genotypes

showed substantial variation in shoot dry matter,

water use, stomatal conductance, leaf tempera-

ture, transpiration efficiency, carbon isotope dis-

crimination (D13C), relative water content (RWC)

and osmotic potential, determined at pre-flower-

ing vegetative stage. Moisture deficits decreased

water usage and consequently shoot dry matter

production. RWC, osmotic potential, stomatal

conductance and D13C were lower, whereas leaf

temperature and transpiration efficiency were

higher in stressed plants, probably due to re-

stricted transpirational cooling induced by stoma-

tal closure. Furthermore, differences in stomatal

conductance, leaf temperature, D13C and transpi-

ration efficiency characterized genotypes that

were physiologically more adapted to water

deficit conditions. Correlation analysis also

showed relatively strong relationships among

these variables under well watered conditions.

The drought tolerant genotypes, ILB-938/2 and

Melodie showed lower stomatal conductance

associated with warmer leaves, whereas higher

stomatal conductance and cooler leaves were

observed in sensitive lines (332/2/91/015/1 and

Aurora/1). The lower value of D13C coupled with

higher transpiration efficiency in ILB-938/2, rel-

ative to sensitive lines (Aurora/1 and Condor/3),

is indeed a desirable characteristic for water-

limited environments. Finally, the results showed

that stomatal conductance, leaf temperature and

H. R. Khan (&) � T. J. HockingSchool of Applied Sciences, University ofWolverhampton, Wulfruna Street, WolverhamptonWV1 1SB, UKe-mail: H.Khan6@wlv.ac.uk

W. LinkInstitute of Agronomy and Plant Breeding, vonSiebold Straße 8, 37075 Gottingen, Germany

F. L. StoddardDepartment of Applied Biology, University ofHelsinki, P.O. Box 27, 00014 Helsinki, Finlande-mail: frederick.stoddard@helsinki.fi

123

Plant Soil (2007) 292:205–217

DOI 10.1007/s11104-007-9217-5

D13C are promising physiological indicators for

drought tolerance in faba bean. These variables

could be measured in pot-grown plants at ade-

quate water supply and may serve as indirect

selection criteria to pre-screen genotypes.

Keywords Carbon isotope discrimination �Leaf temperature � Stomatal conductance �Transpiration efficiency

Abbreviations

D13C Carbon isotope discrimination

RWC Relative water content

TE Transpiration efficiency

Introduction

Production systems in European agriculture have

been predominantly cereals-based but in the

recent past, economic and environmental devel-

opments have revived interest in grain legumes as

break crops that break cereal disease cycles, fix

nitrogen and yield pulses for feed and food uses

(Plies-Balzer et al. 1995; Robson et al. 2002).

Among the grain legumes, faba bean is adapted to

a broad range of environmental conditions and is

the subject of considerable attention in Europe.

The cultivated area of faba bean in the UK, for

example, has increased by 87% during the last

10 years (DEFRA Statistics 2005). Nevertheless,

yields have remained lower and more variable

than those of cereals. Drought is a major abiotic

constraint responsible for heavy production losses

(Link et al. 1999; Amede et al. 1999; Ricciardi

et al. 2001). Furthermore, the climatic-change

models predict that in many regions of Europe,

drought losses and yield variability of field crops

will increase (Marsh 1996).

Grain legumes respond to drought differently

and express various drought tolerance strategies

(Subbarao et al. 1995). Faba bean is more sensi-

tive to drought than some other grain legumes

including common bean, pea and chickpea

(McDonald and Paulsen 1997; Amede and Schu-

bert 2003). Although genotypic differences in the

response of faba bean to drought have been

documented (Heringa et. al. 1984; Grzesiak et al.

1997; Abdelmula et al. 1999; Amede et al. 1999;

Link et al. 1999), the physiological processes

associated with drought tolerance are less under-

stood than for other crop species.

The development of drought-tolerant cultivars

is essential to improve the yield stability of faba

bean. Plant breeders have considerable capacity

to evaluate breeding material for drought toler-

ance under field conditions based on grain yield at

drought-prone sites (Grzesiak et al. 1997; Link

et al. 1999), but the routine evaluation of drought

tolerance in the field is expensive, prolonged and

often produces variable results due to seasonal

variation. Blum (1984) suggested that genotypes

that show better performance under hostile envi-

ronments generally possess some unidentified

physiological attributes of tolerance to environ-

mental stresses in good conditions. It is impera-

tive for breeding drought resistant cultivars to

identify the specific physiological traits that

improve adaptation to water-limited environ-

ments (Subbarao et al. 1995). So far, progress

using this approach has been slow mainly due to

lack of an efficient screening technique (Wery

et al. 1994; Stoddard et al. 2006). An ideal

screening technique should be non-destructive,

rapid, accurate and able to handle many samples

(Reynolds et al. 1994). Therefore, understanding

the physiological basis of drought tolerance in

faba bean is important to identify traits, which can

be used as indirect selection criteria. The objec-

tives of the present investigation were first, to

determine the magnitude of genetic diversity in

physiological traits related to drought tolerance in

faba bean inbred lines and second, to explore

relationships among potentially useful traits to be

used in breeding programs for drought tolerance.

Materials and methods

The experiments were designed as completely

randomized factorial designs with four replica-

tions. Plants were grown in 4 L plastic pots filled

with 3.0 kg potting mix containing all essential

nutrients. The soil moisture was kept at field

capacity (20% w/w) in all pots by applying water

on alternate days until the induction of moisture

stress treatment. Water use increased with plant

206 Plant Soil (2007) 292:205–217

123

growth. Four inbred lines were used in experiment

1 and six inbred lines in experiment 2 (Table 1).

Lines of diverse genetic backgrounds were chosen

from those that Abdelmula et al. (1999) and Link

et al. (1999) characterized for drought response in

field conditions. Seeds were germinated on moist

Whatman filter paper at 20�C for 72 h in an

incubator. Two radicle-emerged seeds were sown

at 3-cm depth in each pot and were later thinned to

one plant per pot. Plants were not inoculated with

Rhizobium in these experiments. A layer of 3-mm

white polypropylene beads ~1 cm deep was placed

over the soil surface as mulch and reduced evapo-

ration to 5–7 mL day–1 in unplanted pots. Plants

were grown in a growth chamber (1400 Plant Growth

Room, Fisons Environmental Equipment, Lough-

borough, UK). Pots were 15 cm apart and photope-

riod was adjusted to 14 h light and 10 h dark, and the

temperature was 20�C day/10�C night ± 2�C. Pho-

tosynthetic photon flux density (PPFD) was about

300 lmol m–2 s–1 at the canopy level.

Moisture stress was induced in half of the pots

43 days after sowing (experiment 1) or 41 days

after sowing (experiment 2) by decreasing water

application by 2% (w/w) per day to bring the

moisture level down from field capacity (20% w/

w) to moisture stress (2–4% w/w). Pots were

weighed daily and where water use exceeded 2%,

water was applied. The aim was to control the

development of moisture stress to a slow rate that

was kept to a consistent level across accessions.

Water use was estimated from the loss in weight

by weighing the pots at watering. Relative water

content (RWC%) was determined on five leaf discs

of 6 mm diameter each, punched from the upper-

most fully expanded leaf of the main stem, 7–

8 days after the induction of water stress. After the

fresh weight was determined, then the discs were

floated for 20 h at room temperature on deionized

water in a covered Petri dish at light near the

compensation point (10–12 lE m–2 s–1). After

rehydration, the discs were surface-dried using tissue

paper, transferred into glass vial and turgid weight

was obtained. The final dry weight was determined

after drying in a pre-heated oven at 80�C for 24 h.

RWC was calculated according to Turner (1981).

Water relation measurements were taken

between 1100 and 1300 hours. Stomatal conduc-

tance was determined 6 days after induction of

water stress using a porometer (Mk 3, Delta- T

Devices, Cambridge, UK) on the abaxial surface

of the leaflets of the uppermost fully expanded

leaf. Leaf temperature readings of the three

leaflets of a fully illuminated youngest expanded

leaf were taken with a non-contact infrared

thermometer (Raynger ST60 ProPlus, Raytek,

Santa Cruz, CA, USA), held at an appropriate

angle about 10 cm above the leaf, focussing the

laser point in the centre of the leaflet. To measure

osmotic potential (wp), a leaflet from the same

leaf used for RWC was excised directly into a

plastic microcentrifuge tube and immediately

frozen in liquid nitrogen. The tubes were later

transferred to –30�C and kept frozen until mea-

surements. The frozen leaf material was allowed

to thaw at room temperature for 30 min and sap

was extracted using a 2.5-mL syringe. A filter

paper disc was placed in the sampling chamber of

a vapour pressure osmometer (model 5100C;

Wescor, Logan, UT, USA) and 8 lL sap was

applied to saturate the disc to measure osmotic

potential. At the end of 10-day drying cycle, the

youngest expanded leaf (usually the one following

the leaf chosen for osmotic potential analysis) was

collected from water stressed and well watered

plants and dried in an oven at 80�C for 24 h for

carbon isotope determination. The stable carbon

isotope composition in the leaf tissue was deter-

mined by elemental analyser isotope ratio mass

spectrometry (Iso-analytical Ltd., Sandbach,

Cheshire, UK) using standard procedures and

techniques. Carbon isotope discrimination (D13C)

was calculated according to Farquhar and Rich-

ards (1984), assuming an isotope composition for

Table 1 Names and origins of accessions used to evaluatedrought response in faba bean

Accession Origin Experiment

332/2/91/015/1 Interpoola 2Aurora/1 Sweden 1BB686WN/1 Germany 2Condor/3 Germany 1ILB 2282/2 Ecuador 2ILB 938/2 ICARDA 1, 2Melodie France 1, 2Victor/2 Netherlands 2

a Inbred line derived from Mediterranean · CentralEuropean cross

Plant Soil (2007) 292:205–217 207

123

the air (dair) of –8&. At the end of the experi-

ments, plants were cut off at soil level and dried in

an 80� oven for 24 h for dry matter determination.

Transpiration efficiency was calculated as unit

weight of dry matter produced per unit weight of

water transpired. The data recorded during the

course of the experiments were subjected to

analysis of variance and pairs of means were

compared using least significant differences

(LSD) at 5% level of probability (P < 0.05).

Results

Experiment 1

Shoot dry matter and water use

Shoot dry matter accumulation and total water

use varied significantly among accessions and as

expected, shoot dry matter was significantly

higher in non-stress conditions than in stressed

plants. ILB938/2 was fast growing, producing

most shoot dry matter, followed by Melodie,

while Condor/3 was relatively slow growing

(Fig. 1). Reduction in shoot growth due to mois-

ture stress was observed in all accessions but it

was more obvious in fast growing lines such as

ILB938/2 and Melodie. The trend in water use

among accessions in the water deficit treatment

was similar to that observed at adequate water

supply (Fig. 1). However, the differences among

accessions in total water use were greater under

well-watered conditions compared to the water

stress treatment. ILB 938/2 consumed signifi-

cantly more water than Condor/3 (Fig. 1).

Relative water content, stomatal conductance and

leaf temperature

Induction of water stress resulted in a consider-

able decline in RWC (from 83% in well-watered

plants to 68% in stressed ones, Table 2) and

osmotic potential (from –0.89 to –1.0 MPa,

Table 2) along with decreases in stomatal con-

ductance (145–53 mmol m–2 s–1, Table 3) and

increases in leaf temperature (18.9�–20.2�, Ta-

ble 3), showing that the implementation of mois-

ture deficit successfully put the plants under

stress.

The differences in RWC among accessions

were significant at adequate moisture supply and

also under stress conditions. Condor/3 maintained

the highest RWC values (Table 2). At sufficient

soil moisture Aurora/1 had the lowest RWC

(81.5%) compared with ILB 938/2 (82.8%). In

both stress and non-stress conditions, Aurora/1

had significantly lower osmotic potential than

ILB 938/2 and Condor/3 (Table 2). The geno-

type · stress interaction was not significant for

osmotic potential.

Genotypic variability in stomatal characteris-

tics was found when moisture supply was not

restricted and the accessions responded differ-

ently to the stress (Table 3). ILB 938/2 and

Melodie had comparably low stomatal conduc-

tance under well-watered conditions and rela-

tively high conductance in the stressed condition.

Condor/3, in contrast, went from second-highest

conductance in well-watered conditions to lowest

when stressed.

Condor/3 Aurora/1 Melodie ILB938/2

0

2

4

6

8

10

Stress

No stress

Sho

ot d

ry m

atte

r (g

)w

ater

use

(L)

Condor/3 Melodie Aurora/1 ILB938/20

1

2

3

a

b

Fig. 1 a Shoot biomass per plant (g) and b total water useper plant (L) of four faba bean accessions. Plants in thewater stress treatment experienced increasing moisturedeficit from day 43, whereas well-watered plants weremaintained at field capacity and all plants were harvested53 days after sowing

208 Plant Soil (2007) 292:205–217

123

Leaf temperature by contrast, showed no

significant difference among the lines in the stress

conditions, but significant difference under the

non-stress conditions, with Aurora/1 and Condor/

3 having significantly lower leaf temperatures

than ILB 938/2 and Melodie (Table 3).

Carbon isotope discrimination and transpiration

efficiency

Carbon isotope discrimination varied significantly

among accessions and was also affected by stress

levels, while the accession · stress interaction was

not significant (Table 4). Aurora/1 and Condor/3

had equally high values of D13C (23.3 · 10–3),

while ILB 938/2 had the lowest (21.6 · 10–3). The

values of D13C were significantly lower in water

stressed plants but the ranking of accessions was

not altered by water regimes.

Transpiration efficiency increased when plants

suffered from moisture stress but the acces-

sion · stress interaction was not significant. Melo-

die and ILB-938/2 were more efficient in water

use and maintained significantly higher transpira-

tion efficiency then Aurora/1 and Condor/3,

regardless of water stress levels (Table 4).

Relationships among physiological traits

Correlation analysis showed some strong rela-

tionships among physiological traits. As expected,

shoot dry matter and transpiration efficiency were

Table 2 Effect of water deficit on relative water content (%) and osmotic potential (-MPa) of four faba bean accessions

Genotypes Relative water content (%) Osmotic potential (-MPa)

Stress No stress Stress No stress

Aurora/1 68.1 81.5 1.02 0.83Condor/3 70.2 83.4 0.98 0.76ILB 938/2 66.4 82.8 0.91 0.78Melodie 68.3 82.7 1.09 0.81Mean 68.2 82.6 1.00 0.79LSD (P £ 0.05)Genotype 1.0** 0.04**Stress 0.7** 0.03**Genotype · stress 1.4** NS

Relative water content and osmotic potential was recorded 8 and 9 days after the induction of water stress, respectively

NS non significant

** P < 0.01

Table 3 Stomatal conductance (mmol m–2 s–1) and Leaf temperature (�C) as influenced by water deficit in four faba beanaccessions

Genotypes Stomatal conductance (mmol m–2 s–1) Leaf temperature (�C)

Stress No stress Stress No stress

Aurora/1 58 176 20.3 18.3Condor/3 30 152 20.2 18.6ILB 938/2 56 125 20.1 19.3Melodie 68 127 20.2 19.4Mean 53 145 20.2 18.9LSD (P £ 0.05)Genotype 13** NSStress 10** 0.3**Genotype · stress 19** 0.6*

Measurements were taken 6 days after the induction of water stress

NS non significant

*, ** P < 0.05, 0.01, respectively

Plant Soil (2007) 292:205–217 209

123

negatively correlated with D13C, showing parallel

lines for the two water treatments (Fig. 2). In the

well-watered plants, leaf temperature and specific

leaf weight exhibited strong negative correlation

with D13C (Fig. 3), while significant positive rela-

tionships were observed between stomatal con-

ductance and D13C. In stress conditions these

relationships were generally non-significant.

Experiment 2

Relative water content and osmotic potential

Induction of water stress was again successful at

reducing RWC, osmotic potential (Table 5). ILB

938/2 and Melodie had significantly higher RWC%

than Victor/2 and line 332/2/91/015/1, which con-

sistently was the lowest (Table 5). Differences in

osmotic potential were also found at adequate

moisture supply, and Melodie and 332/2/91/015/1

had lower osmotic potential than Victor/2 and BB

686WN/1 (Table 5). Moisture stress resulted in a

significant decline in osmotic potential in all entries

except ILB938/2; the highest decrease (0.17 MPa)

was recorded in Victor/2.

Transpiration efficiency, stomatal conductance

and leaf temperature

Transpiration efficiency based on shoot dry mat-

ter accumulation varied significantly among

accessions (Fig. 4). ILB 938/2 was once again

most efficient in water use (4.03 g L–1), while the

lowest value of transpiration efficiency was

Table 4 Effect of water stress on carbon isotope discrimination (D · 10–3) and transpiration efficiency (g L–1) in four fababean genotypes

Genotypes Carbon isotope discrimination (D · 10–3) Transpiration efficiency (g L–1)

Stress No stress Stress No stress

Aurora/1 21.4 23.3 2.99 2.82Condor/3 21.7 23.3 2.73 2.61ILB 938/2 20.1 21.6 3.30 3.16Melodie 20.5 22.4 3.37 3.15Mean 20.9 22.7 3.10 2.94LSD (P £ 0.05)Genotype 0.4** 0.13**Stress 0.3** 0.09**Genotype · stress NS NS

Plants were harvested 53 days after sowing and subjected to water deficit for the last 10 days

NS non significant

** P < 0.01

15

17

19

21

23

25

2 3 4 5 6 7 8 9

Car

bon

isot

ope

disc

rimin

atio

n C

arbo

n is

otop

e di

scrim

inat

ion

Stress

Well-watered

y = 24.3 - 0.57 x

r = - 0.68**

y = 25.9 - 0.48 x

r = - 0.64**

15

17

19

21

23

25

2.0 2.5 3.0 3.5 4.0

Transpiration efficiency

Shoot dry matter

y = 27.6 - 2.15 x

r = - 0.81**

y = 28.9 - 2.08 x

r = - 0.84**

a

b

Fig. 2 Relationship of carbon isotope discrimination(D · 10–3) with a shoot dry matter production (g) and btranspiration efficiency (g L–1). Plants in the stresstreatment received restricted supply of water for the last10 days whereas the other plants were well watered

210 Plant Soil (2007) 292:205–217

123

recorded in line 332/2/91/015/1. The difference in

transpiration efficiency between water treatments

was not significant.

Induction of water stress reduced stomatal

conductance (Table 6) along with increasing leaf

temperature. At adequate moisture supply,

ILB938/2 and Melodie had significantly lower

stomatal conductance than lines 332/2/91/015/1,

ILB2282/2 and Victor/2 (Table 6). The decline of

stomatal conductance in ILB 938/2 in response to

water stress was much more rapid than in 332/2/

91/015/1 (Fig. 5). The decrease in stomatal con-

ductance was significantly higher in BB686WN/1,

ILB 938/2 and Melodie, compared to 332/2/91/

015/1 and ILB2282/2.

The entries had significant variation in leaf

temperature even when moisture supply was

adequate (Table 6). ILB938/2 had warmer leaves

than 332/2/91/015/1 and ILB2282/2. Although

moisture stress raised the leaf temperature in all

genotypes, lines 332/2/91/015/1, ILB2282/2 and

BB686WN/1 showed less change than ILB938/2.

Relationships among physiological traits

In this experiment, stomatal resistance under

well-watered conditions was again positively cor-

related with transpiration efficiency and leaf

temperature, and unlike experiment 1 with

RWC (data not shown). At high water supply,

leaf temperature had a significant and positive

correlation with leaf RWC (not significant in

experiment 1) and, as before, transpiration effi-

ciency (Fig. 6). Transpiration efficiency was pos-

itively correlated with RWC but the slope was not

significantly different between water treatments

(data not shown).

Discussion

The present study showed wide variation in the

transpiration characteristics of faba bean lines.

These traits were correlated with each other, to a

greater or lesser degree, and the variation in some

traits in well-watered conditions was indicative of

the variation in drought response in the field.

The chosen faba bean lines varied considerably

in shoot dry matter production and water use

pattern. The imposed reduction in water supply

was sufficient to induce moisture stress, which

consequently reduced plant growth. After 10 days

of restricted water supply, leaves had wilted,

particularly in Aurora/1 and line 332/2/91/015/1.

Bond et al. (1994) reported that the faba bean

18

20

22

24

26

50 100 150 200 250

Stomatal conductance

Car

bon

isot

ope

disc

rimin

atio

nC

arbo

n is

otop

e di

scrim

inat

ion

Car

bon

isot

ope

disc

rimin

atio

n

y = 20.2 + 0.017 *X

r = 0.62*

16

18

20

22

24

26

18 19 20 21 22

Leaf temperature

y = 38.5 - 0.79 x

r = - 0.74**

18

20

22

24

2.0 2.5 3.0 3.5 4.0

Specific leaf weight

y = 8.36 - 0.23 x

r = - 0.78**

a

b

c

Fig. 3 Relationship of carbon isotope discrimination(D · 10–3) with a stomatal conductance (mmol m–2 s–1), bleaf temperature (�C), and c specific leaf weight (mg cm–2)in well watered plants

Plant Soil (2007) 292:205–217 211

123

Table 5 Effect of water deficit on relative water content (%) 7 days after induction of water stress and osmotic potential(-MPa) 8 days after induction in six faba bean genotypes

Genotypes Relative water content (%) Osmotic potential (-MPa)

Stress No stress Stress No stress

332/2/91/015/1 61.1 68.2 0.90 0.81BB 686 WN/1 73.1 77.6 0.86 0.75ILB 2282/2 68.3 77.3 0.86 0.77ILB 938/2 74.2 77.7 0.79 0.78Victor/2 70.3 73.9 0.91 0.74Melodie 71.0 78.6 0.91 0.82Mean 69.6 75.6 0.87 0.78LSD (P £ 0.05)Genotype 2.3** 0.04**Stress 1.3** 0.02**Genotype · stress NS 0.05**

NS non significant

** P < 0.01

332/2/91/015/1 ILB2282/2 Victor/2 Melodie

Tra

nspi

ratio

n ef

ficie

ncy

BB686 WN ILB938/2

0

1

2

3

4

Fig. 4 Variation intranspiration efficiency(g L–1) among six fababean inbred lines. Plantswere harvested at pre-flowering stage, 50 daysafter sowing

Table 6 Stomatal conductance (mmol m–2 s–1) 5 days after induction of water stress and leaf temperature (�C) 6 days afterinduction in six faba bean genotypes

Genotypes Stomatal conductance (mmol m–2 s–1) Leaf temperature (�C)

Stress No stress Stress No stress

332/2/91/015/1 79 175 18.7 16.2BB 686 WN/1 35 119 18.7 17.0ILB 2282/2 67 141 18.2 16.9ILB 938/2 39 99 19.2 17.6Victor/2 45 137 18.9 17.4Melodie 41 106 18.9 17.1Mean 519 129 18.8 17.0LSD (P £ 0.05)Genotype 19** 0.3**Stress 11** 0.2**Genotype · stress NS 0.4**

NS non significant

** P < 0.01

212 Plant Soil (2007) 292:205–217

123

plant requires a large amount of water to retain

turgor in its fleshy leaves, making it relatively

sensitive.

In our experiments, plants were at a consistent

vegetative stage (pre-flowering) when water stress

was induced. We found inherent differences in

growth rate among the lines, so care was taken to

induce an even moisture stress in the pots by

monitoring water use daily and applying water if

the water use was > 2% w/w. Pot experiments

have been successfully used previously with

legumes to facilitate identification of drought

tolerant breeding materials (Amede et al. 1999).

Balko et al. (1995) reported that the rank order of

faba bean lines subjected to moisture stress in

pots was reproducible and strongly correlated

with their relative yield in field trials. Ricciardi

et al. (2001) used controlled environments to

effectively induce water stress in faba bean and

it was also possible to simulate stress of different

intensities for evaluation of breeding material for

moisture deficit tolerance.

In the present study, numerous physiological

traits that can contribute towards yield under

drought conditions were assessed in entries of

diverse genetic backgrounds, representing a wide

range of origins. The accessions differed in their

water usage and the trend in water use in the

moisture deficit treatment was very similar to that

observed at adequate water supply. Furthermore,

the differences among genotypes were more obvi-

ous and consistent in the well-watered conditions

than in the water stress treatment. ILB 938/2 and

Melodie exhibited high transpiration efficiency in

both experiments indicating that these are more

efficient in water use. These results are in agree-

ment with the field observations, which character-

ized ILB 938/2 and Melodie as drought-tolerant,

with high relative yield under simulated drought

conditions (Abdelmula et al. 1999; Tolera and

Link 2002). In contrast, Aurora was sensitive to

0Day 1 Day 3 Day 5

50

100

150

200S

tom

atal

con

duct

ance

332/2/91/015/1 Stressed

332/2/91/015/1 Well watered

ILB 938/2 Stressed

ILB 938/2 Well watered

Fig. 5 Stomatal response of ILB 938/2 and 332/2/91/015/1to the development of moisture stress or well watered inpots under controlled condition (Experiment 2). Stomatalconductance measurements are in mmol m–2 s–1

123.0 3.5 4.0 4.5

14

16

18

Transpiration efficiency

y = 11.3 + 1.3 x

r = 0.66**

12

14

16

18

60 70 80 90Relative water content

Leaf

tem

pera

ture

Le

af te

mpe

ratu

re

y = 10.9 + 0.068 x

r = 0.59**

a

b

Fig. 6 Relationship of leaf temperature (�C) with atranspiration efficiency (g L–1), and b relative watercontent (%) in well watered plants. Transpiration effi-ciency is based on shoot biomass of plants, harvested atpre-flowering stage

Plant Soil (2007) 292:205–217 213

123

drought in field conditions (Amede et al. 1999) as

well as in our experiments.

Carbon isotope composition has been used to

determine the genotypic and environmental

responses of water use efficiency in several

legume species, including lentil (Matus et al.

1996), alfalfa (Johnson and Rumbaugh 1995),

common bean (Zacharisen et al. 1999) and chick-

pea (Khan et al. 2004) and now faba bean.

Our experiments showed considerable genetic

variation for D13C in the leaf material among faba

bean genotypes. The drought tolerant accessions,

ILB 938/2 and Melodie, had significantly lower

D13C than sensitive varieties such as Aurora/1 and

332/2/91/015/1 at adequate moisture supply.

Selection for low D13C has been proposed as a

method to select for improved water use effi-

ciency in breeding programs for C3 crop species

(Johnson and Rumbaugh 1995) and the present

data support this. Correlation analysis conducted

using genotypic means demonstrated that D13C

was negatively associated with transpiration effi-

ciency, stomatal resistance and leaf temperature.

Similarly, lentil showed genetic variation for D13C

(Matus et al. 1996), which was strongly and

negatively correlated with water use efficiency

(Johnson et al. 1995). Water stress also led to

reduced D13C and increased transpiration effi-

ciency in faba bean, indicating that drought

altered the balance between the stomatal con-

ductance and internal leaf capacity for photosyn-

thesis, which determine the internal leaf CO2

concentration and D13C, as observed in alfalfa

(Johnson and Tieszen 1994). The utility of D13C

for selection in a plant breeding program is

strongly enhanced by the consistency of genotypic

ranking for different water regimes (Hall et al.

1992). Our results suggest that D13C may be a

useful parameter for selecting faba bean geno-

types that use water efficiently under water-

limited conditions. Additional data concerning

the physiological basis of D13C response at

restricted moisture supply and association with

grain yield in faba bean, are required to confirm

that selection for low D13C would lead to signif-

icant gains in water use efficiency.

Variation in drought response between faba

bean lines was mainly associated with dehydra-

tion avoidance through stomatal control (Amede

et al. 1999). Nerkar et al. (1981) found consider-

able differences in water loss and transpiration in

faba bean genotypes having contrasting stomatal

features, and suggested that lower transpiration

rate would contribute to drought tolerance. Fur-

thermore, excessive water use in the drought-

sensitive faba bean cultivar Adriewaalse from

Netherlands was associated with its higher tran-

spiration, while L-7, a breeding line of Mediter-

ranean background was modest in water use and

hence drought tolerant (Amede et al. 1999).

The extent of physiological variability in the

stomatal characteristics among genotypes is of

primary importance, as it constitutes an efficient

control mechanism of water loss that can be used in

breeding programmes (Tanzarella et al. 1984).

Our results revealed marked variation in stomatal

conductance among inbred lines, with lower sto-

matal conductance and higher transpiration effi-

ciency in ILB938/2 and Melodie at adequate water

supply compared with lines 332/2/91/015/1 and

Victor/2. This confirms the physiological signifi-

cance of lower stomatal conductance in regulating

the water loss from leaves. Stomatal characteristics

vary widely in faba bean (Ricciardi 1989). Fre-

quency and size of stomata also differed between

drought-resistant faba bean cultivar Gobo and

sensitive cultivar Victor (Grzesiak et al. 1997).

Therefore, lower stomatal conductance seems a

useful physiological attribute for evaluation of

breeding material for superior water use efficiency.

We found that leaf temperature at adequate

water availability differentiated genotypes for

drought response. In the presence of drought

stress, when the stomatal conductance data indi-

cated that the stomata were largely closed, there

was little variation in leaf temperature. In well-

watered conditions, however, warmer leaves were

associated with low stomatal conductance and

high-transpiration efficiency, which are favourable

attributes for drought adaptation. Idso et al. (1981)

used the increase in average canopy temperature

following drought stress as a key component of a

crop water stress index and O’Neill et al. (2006)

considered leaf temperature as a potential indica-

tor of plant water stress, since increasing plant

water deficit leads to stomatal closure, decreases

transpirational cooling and consequently increases

leaf temperature.

214 Plant Soil (2007) 292:205–217

123

The application of infrared thermometry to

irrigated wheat demonstrated that canopy tem-

perature measurably decreased as stomatal con-

ductance increased (Fischer et al. 1998). Wheat

cultivars with the warmest canopy temperatures

under well watered conditions not only had the

lowest leaf conductance and the lowest seasonal

water use under normal irrigation practices but

they also had the most favourable yield response

when subjected to water deficit conditions (Pinter

et al. 1990). The difference between leaf temper-

ature and air temperature was linearly related to

stomatal conductance in spring wheat and there-

fore, it might serve as surrogate for stomatal

conductance (Amani et al. 1996). Pea cultivars

showing higher transpiration rate had higher

stomatal density that effectively reduced canopy

temperature due to transpirational cooling (San-

chez et al. 2001). Hence, non-invasive approaches

using infrared thermometry to classify genotypes

according to canopy temperature appear feasible

for the efficient screening for water use charac-

teristics and tolerance to drought (Pinter et al.

1990).

This study demonstrates the potential useful-

ness of leaf temperature data for identifying the

water use characteristics of faba bean genotypes.

A simple hand-held infrared thermometer can be

used for measuring leaf temperature relatively

easily and precisely in faba bean, owing to its

broad leaves. Furthermore, genotypic differences

can be detected in controlled environments,

reducing error due to subtle weather conditions.

Although it is possible to evaluate leaf tempera-

ture differences during the vegetative stage at

different water regimes, our results show that

measurements should be taken at ample soil

water to safeguard against differential rates of

water use confounding the results. Similarly,

Singh and Kanemasu (1983) observed leaf tem-

perature differences of 5.0�C in pearl millet

genotypes under well-watered conditions,

whereas differences in leaf temperature were less

in water stressed plants, and they suggested that

canopy temperature in a non-stressed environ-

ment would be a valid criterion for screening

genotypes for their yield stability.

Cost is important when considering a useful

indirect selection criterion for drought tolerance.

Determination of D13C is expensive and the non-

contact infrared sensing system offers exciting

possibilities for cost reduction (Fischer et al.

1998). Canopy temperature measurement using

the infrared thermometer has been recommended

as the physiological trait of choice for evaluating

cereal drought response, for the obvious reasons

that it is very rapid, giving instant measurements,

and can be measured on single leaves inexpen-

sively and quickly, unlike many other physiolog-

ical parameters (Reynolds et al. 1994). The

infrared thermometry may have potential utility

in water-limited environments because relatively

small differences in plant transpiration are gen-

erally translated into canopy temperature differ-

ences of several degrees (Pinter et al. 1990). Our

results confirm that leaf temperature measure-

ments show promise for identifying desirable

traits among genotypes in breeding nurseries,

preferably at adequate water supply, and infrared

thermometers may be used to pre-screen for

physiological responses related with drought tol-

erance under controlled conditions prior to the

execution of extensive yield trials.

Conclusion

This study has shown genotypic diversity for

drought-associated physiological traits in a di-

verse set of European and Mediterranean faba

bean inbred lines. Furthermore, the variation in

stomatal conductance, leaf temperature, transpi-

ration efficiency and D13C characterized geno-

types that are physiologically more adapted to

water deficits. A correlation analysis showed

strong relationships among these variables. Inter-

estingly, drought tolerant genotypes, ILB-938/2

and Melodie showed lower stomatal conductance

than drought-sensitive lines (332/2/91/015/1 and

Aurora/1), probably a drought avoidance mech-

anism for minimizing water loss through re-

stricted transpiration. The cooler canopy of

sensitive lines relative to ILB-938/2 and Melodie

further explained genotypic response to stomatal

characteristics, as wide and open stomata may

enhance transpirational cooling. The lower value

of D13C associated with higher transpiration

efficiency in ILB-938/2 would be regarded as a

Plant Soil (2007) 292:205–217 215

123

desirable characteristic in breeding programs for

water-limited environments.

Finally, it appears that stomatal conductance;

leaf temperature and D13C are potential indica-

tors for drought tolerance. Although these char-

acteristics were measured in pot-grown plants at a

pre-flowering vegetative stage, there is a possibil-

ity of exploiting these differences in a breeding

program to develop lines less susceptible to

drought stress. The study was conducted within

the framework of a faba bean breeding for

sustainable agriculture program geared mainly

towards identifying genotypes with physiological

attributes of superior drought adaptation. These

results obtained in pot experiments supported by

pre-existing field data will help assess the genetic

determination of differences in drought tolerance.

Acknowledgements This work was carried out with thefinancial support of EU that was provided under projectQLK5-CT-2002-02307 ‘‘Faba bean Breeding forSustainable Agriculture’’, acronym EUFABA. Technicalassistance of Robert Hooton and Fiona Bowers for thiswork is gratefully acknowledged.

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