Sugar Beet Seed Institute, Karadj, Iran

Effect of Water-Deficit Stress on Germination and Early Seedling Growth in Sugar

Beet

S. Y. Sadeghian and N. Yavari

AuthorsÕ address: Dr S. Y. Sadeghian (corresponding author; e-mail: [e-mail]) and N. Yavari, Sugar Beet Seed
Institute, PO Box 31585-4114, Karadj, Iran

With 2 figures and 3 tables

Received May 9, 2003; accepted August 29, 2003

Abstract

Sugar beet progeny lines screened for both high water use
efficiency and high sugar yield under drought stress
conditions in the field were assessed for the rate of seed
germination and early seedling growth in water deficit
stress, induced by mannitol solutions. Seeds of nine
different sugar beet progeny lines were grown in three
experimental conditions using filter paper, perlite and water
agar as substrate. Three levels of 0.0, 0.2 and 0.3 m
mannitol concentrations were applied in each experiment.
A factorial design was used with three replications.
Germination percentage was determined in all experiments.
Seedling growth parameters such as cotyledon fresh weight,
cotyledon dry weight, root fresh weight, root dry weight
(RDW) and root length (RL) were measured experiment-
ally. Abnormality was only recorded in the filter paper
experiment. The results showed that drought stress could
be simulated by mannitol solution and significant differ-
ences were found between stress levels for seedling charac-
teristics. Distinct genetic variances were found among
progeny lines with respect to germination and early seedling
growth characteristics, except for cotyledons and RDW.
Seedling growth and germination rates severely declined at
the highest concentration of mannitol. The rate of abnor-
mality was increased progressively at the germination stage
with an increase in mannitol concentration but it was more
pronounced in the drought-susceptible progeny lines. The
highest values of relative germination % and relative
growth % of RL were obtained for the most tolerant line.
In conclusion, seedling characteristics, in addition to other
physiological components involved in the seed germination
process under specific stress conditions, may be considered
for breeding purposes.

Key words: germination — in vitro — line-seedling-
characteristics — mannitol — stress — sugar beet

Introduction

Spring-sown sugar beet crop in semi-arid condi-
tions needs to be irrigated. This coincides with the

full growing period of other economically import-
ant crops such as wheat. Each year the sugar beet
seedlings suffer due to lack or delay of irrigation
that hampers plant growth, affects final plant
density and ultimately the root yield of the crop.

Seed germination is the most important seed

quality, and germination under laboratory condi-
tions and field establishment are closely related
characteristics that are influenced by genetic (vari-
etal), environmental (location and year) and seed-
processing (treatments) effects (Apostolides and
Goulas 1998). Studies on abiotic stress tolerance in
sugar beet have been undertaken for the identifi-
cation of physiological and environmental factors.
Germination percentage of sugar beet measured
under standard conditions correlated with the
seedling establishment in the field under stress
conditions (Durrant et al. 1985, Durrant and
Gummerson 1990).

Unfavourable

germination

conditions such as low temperature and water
stress have serious impacts on the results of vigour
tests and plant establishment of diploid and trip-
loid sugar beets in the field (Van Swaaij et al. 2001).
Delayed seedling emergence of sugar beet was
found to occur as a result of water stress and
increasing salinity (Ayers 1952). The effects of
abiotic stresses were studied on young sugar beet
seedlings in the laboratory using osmotic agents
such as mannitol, polyethylene glycol (PEG) and
salts in Petri dishes containing Whatman filter
paper (Ghoulam and Fares 2001).

Small differences in the concentration of NaCl

did not change number of germinated seeds but
greatly affected water uptake and seedling growth
(Durrant et al. 1974). Kaffka and Hembree (1999),
investigating the effect of seed priming on emer-
gence rate and seedling growth of sugar beet in

J. Agronomy & Crop Science 190, 138—144 (2004)
2004 Blackwell Verlag, Berlin
ISSN 0931-2250

U.S. Copyright Clearance Centre Code Statement:

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saline soils, reported a similar rate of germination
for primed seeds in saline plots and non-primed
seeds in plots with low electric conductivity of soil
(ECe). This practice is certain to become a seed-
enhancing requirement for crop production under
stressed conditions.

Nevertheless, breeders assume that there is con-

siderable variability in abiotic stress tolerance in
sugar beet germplasm. Hanson and Wyse (1982)
evaluated sugar beet, fodder beet and Beta mari-
tima

under increasing salinity conditions, and

concluded that salinization increased betaine levels
of roots and shoots two- to three-fold. Beta
maritima

accessions accumulated 40 % less betaine

in shoots than other progeny lines. Studies on
abiotic stress tolerance in sugar beet have been
undertaken for the identification of physiological
and environmental factors. Productivity and ger-
mination are linked to key properties of varieties
used by beet growers and tend to decide the
ultimate crop yield (Tugnoli and Bettini 2001).
Significant variations were found for total dry
matter, root yield, sugar yield, juice purity and
impurities of roots among sugar beet, fodder beet
and red beet when exposed to various periods of
drought stresses (Sadeghian et al. 2000).

The possibility of growing plants in controlled

conditions of greenhouse and laboratory are of
great convenience for the evaluation of genetic
traits. In many crops, seed germination and early
seedling growth are the stages most sensitive
to environmental stresses (Cook 1979). Foolad
and Lin (1999) studied the germination response
of tomato accessions in Petri dishes containing
agar media treated either with 0 mm NaCl (non-
stress and cold-stress treatments) or 150 mm
NaCl + 15 mm CaCl

2

(for salt-stress treatment).

They concluded that some of the same genetic and
physiological parameters contributing to rapid seed
germination under non-stress conditions could also
facilitate rapid seed germination under stress.
Experiments performed at germination and seed-
ling emergence stages of sugar beet under various
temperatures and NaCl concentrations indicated
that the effect of salinity on seedling emergence was
increasingly inhibitory as temperature increased
from 10–15

C to 25 and 35 C (Mahmoud and

Hill 1981). Small differences in the concentration of
NaCl did not change the rate of germinated seeds
but greatly affected water uptake and seedling
growth (Durrant et al. 1974).

The common approach in breeding for drought

tolerance of crops is to select for drought tolerance

components. As several of these components are
difficult to measure, indirect selection should be
applied (Visser 1994). Screening for drought toler-
ance has been reported in vegetable crops using
mannitol as the stress agent (Grezesiak et al. 1996).
Simulated drought condition with mannitol solu-
tion was used to estimate drought resistance in
18 cultivars of field been (Vicia faba), soya bean
(Glycine max), field pea (Pisum sativum), and lupin
(Lupinus albus). The results support the varietal
differences in seed germination and seedling growth
parameters. Osmotic adjustment in tolerant plant
helps maintain leaf metabolism and root growth at
relatively low leaf water potentials by controlling
turgor pressure in the cells. The trait can be assayed
easily by measuring growth rate of seedlings in
PEG solution (Morgan and Condon 1986). More-
over, tolerance ratings under laboratory conditions
were consistent with ratings of the effects of soil
drought on yield in the field experiments, suggest-
ing that nutrient solution containing mannitol
could be used in screening for drought tolerance
in growing seedlings.

Water deficit is one of the main limiting factors

of sugar beet production in arid and semi-arid
lands, therefore breeding programmes should be
explicitly

directed

towards

development

of

drought-tolerant varieties. We report the effects of
an increasing stress agent (mannitol concentra-
tions) on the germination and seedling growth in
sugar beet lines, with an aim to choose an
evaluation procedure for the identification of sugar
beet tolerant to water stress at germination and
early growth phase.

Materials and Methods

Nine sugar beet progeny lines – one very drought tolerant,
four tolerant, two moderate, one rather sensitive and one
sensitive (S

1

and S

2

families) – were previously identified

(Sadeghian et al. 2000) under field condition for their good
yield performances under drought conditions (Table 1).
Seeds of these progeny lines were produced in the same seed
production region, and were graded, standardized, washed,
sterilized and dried at room temperature prior to the
experiments. Germination tests were conducted in three
experiments consisting of filter paper (ISTA standard seed
testing conditions and at 22 ± 2

C), box containing

perlite (greenhouse conditions with night/day temperature
fluctuations from 15 to 23

C), and 0.8 % water-agar

medium in plates (in vitro growth conditions at 25 ± 2

C).

Stress treatments were performed at 0.0 (control), 0.2 and
0.3 m concentrations of mannitol approximately corres-
ponding to

)1, )5 and )7 bar osmotic pressures. In the

greenhouse experiment, boxes (37

· 55 · 13 cm) were

Effect of Water-Deficit Stress on Germination and Early Seedling Growth

139

watered with 1/2 diluted Hoagland solution (Hoagland and
Arnon 1950) for each different mannitol level. The amount
of this solution was adjusted daily to keep a 2-cm level at
the bottom. A factorial design with three replications was
used for each experiment and each treatment replicate
contained 100 seeds/box except in the agar medium, where
25 seeds were grown individually in culture tubes.

Germination was recorded in all the three experiments,

and percentage of abnormal seedlings (germinated seeds
failed in growing standard roots and shoots) was also
calculated in the filter paper test. Germination percentage
was recorded after 14 days in paper, 24 days in perlite and
28 days in agar medium. Seedling characteristics such as
cotyledon fresh weight (CFW), cotyledon dry weight
(CDW), root fresh weight (RFW), root dry weight
(RDW) and root length (RL) were measured in the water
agar medium. Assessments of seedling characteristics were
made 28 days after sowing. The relative germination was
determined by the following calculation: number of germi-
nated seeds in stress medium/number of germinated seeds
in control medium

· 100 (Smith and Dobrenz 1987).

Similarly, relative growth % for RL, RFW and CFW
was calculated for various progeny lines in the in vitro
experiment.

All measured variables were subjected to analysis of

variance (anova) and SAS software was used for the
correlation analysis (SAS Institute, Inc. 1996). Sugar beet
lines were compared for germination %, abnormal seedling
% and relative growth % of seedling characteristics.

Results and Discussion

Records were made on the number of germinated
seed in paper, perlite, and agar medium and of
seedling growth characteristics under in vitro con-
dition. Analysis of variance showed significant
effects of mannitol treatments and progeny lines
(P < 0.001) on the rate of germination in the three
experiments (Table 2). Germination was progres-
sively inhibited by the increase in mannitol
concentration. The strongest inhibition occurred
at the second mannitol concentration (0.3 m) par-
ticularly in perlite and water agar medium. The
response of seeds to an increase in mannitol con-
centration varied among progeny lines (Fig. 1a–c).
Drought stress simulated by mannitol and its
effects on all seedling characteristics were signifi-
cant (Table 2). The seedling growth parameters
were significantly affected by an increase in the
osmotic

pressure

of

mannitol

concentrations

(Fig. 2b–d). Except for very small values obtained
for CDW and RDW, significant differences among
the progeny lines (P < 0.01) were obtained for the
seedling growth traits (Table 2). The effect of
decreasing water potential in 0.2 and 0.3 m mann-
itol concentrations resulted in seedlings with less
fresh weight during the processes of germination
and early growth when compared with the control
treatment (Figs 1 and 2). Analysis of variance for
CFW and RFW indicated that stress condition and
progeny lines had significant differences for these
parameters. Stresses decreased water content and

Table 1: Brief descriptions of sugar beet lines evaluated
in mannitol-stressed conditions

Line no.

Identity no.

Seed type

Drought

tolerance

1

7112

mm

S

2

191

MM

RS

3

181

mm

MT

4

436m

MM

MT

5

7233-17

MM

T

6

7233-12

MM

T

7

BP-Karadj

MM

T

8

463-I-BMM

T

9

7219-69

MM

VT

MM, multigerm; mm, monogerm; S, sensitive; RS, rather
sensitive; MT, medium tolerant; VT, very tolerant.

Table 2: Mean squares from analysis of variance of sugar beet lines tested for the germination rate in filter paper,
perlite and agar medium together with seedling growth characteristics of in vitro seedlings at three levels of mannitol
concentrations

Source of
variation

d.f.

Ger.

in paper

Ger.

in perlite

Ger.

in vitro

CFW

CDW

RFW

RDW

RL

Replication

2

6.5679

14.2716

255.864

0.00526

0.00026

0.00023 0.00005

54. 583

Genotype

8

808.9753

***

676.20

***

1873.688

***

0.0429

**

0.0003

0.0011

**

0.00005

117.544

**

Mannitol

2 1901.3827

***

31071.457

***

16775.308

***

0.8719

***

0.00304

***

0.092

***

0.00022

**

4600.513

***

Variety

·

mannitol

16

134.8827

***

230.734

***

399.964

***

0.01356

0.0002

0.0005

0.00005

43.217

Error

52

37.0807

24.989

6438.272

0.7755

0.0088

0.0188

0.00168

1828.935

Ger., germination %; CFW, cotyledon fresh weight; CDW, cotyledon dry weight; RFW, root fresh weight; RDW,
root dry weight; RL, root length.
**P

¼ 0.01, ***P ¼ 0.001.

140

Sadeghian and Yavari

assimilate accumulation in the seedling as a conse-
quence of osmotic pressure induced by mannitol.
Interaction effects between genotype and mannitol
concentration were significant for the germination
rates in all cases, pointing to the fact that differ-
ences in genetic components affecting the germina-
tion response of sugar beet are expressed at early
stages under specific stress conditions. As shown in
Fig. 1a, the germination percentage recorded in
paper was only slightly affected but number of
abnormal seedlings augmented, as the stress was
intensified (Fig. 1d). The differences in germination
percentage of seeds subjected to stress levels were
more detectable in the perlite and water agar
experiments (Fig. 1b,c). Germination of seed in line
nos 2 (191), 1 (7112) and 3 (181) proved to be most
sensitive to water restriction at the higher level of
osmotic potential in the in vitro experiment, but
seeds of line no. 9 (7219) revealed a relatively stable
tolerance in both stress levels (Fig. 1c).

Evaluation of seedling characteristics presented

as a percentage of the control (Fig. 2a–d) showed

a decrease for all growth parameters as stress
levels intensified. The most tolerant seedlings
showed a better biological efficiency (increase in
size and weight) under increased water deficiency,
while a different distribution of biomass in leaves
and roots were noted (Fig. 2b,d). Here again
line no. 9 (7219–69) responded best for relative
germination and relative growth of RL. Various
progeny lines responded differently for the relat-
ive growth of CFW and RFW (Fig. 2b–d). A
positive correlation was found between germina-
tion rates and seedling characteristics in the three
experiments, except for the percentage of abnor-
mal seedling (Table 3). Coefficients of correlation
between seed germination in paper with that of
perlite and agar medium were strongly significant
(P < 0.001). Abnormality always had a negative
correlation with germination rates and other
seedling characteristics like CDW, CFW, RDW,
RL and RFW, indicating that seedling water
potential and growth efficiency have an essential
role in the development of normal plants. Seed

0

10

20

30

40

50

60

70

80

90

7112

191

181

436mo

7233-107 7233-12 BPKaraj

436-I

7219-69

Lines

A

bnor

. %

0

0.2

0.3

0

10

20

30

40

50

60

70

80

90

100

7112

191

181

436mo

7233-107 7233-12 BPKaraj

436-I

7219-69

Lines

GER. % in Paper

0

0.2

0.3

0

10

20

30

40

50

60

70

80

90

100

7112

191

181

436mo

7233-1077233-12 BPKaraj

436-I

7219-69

Lines

GER. % in perlite

0

0.2

0.3

0

10

20

30

40

50

60

70

80

90

100

7112

191

181

436mo

7233-107 7233-12 BPKaraj

436-I

7219-69

Lines

GER. % in Vitro

0

0.2

0.3

(a)

(b)

(d)

(c)

Fig. 1: Nine sugar beet lines examined for germination percentage (GER. %) in paper (a), perlite (b) and in vitro
(c) at 0.0, 0.2 and 0.3 m concentrations of mannitol. The percentage of seedling abnormality (Abnor. %) in the
paper test is also presented (d). Error bars represent standard error

Effect of Water-Deficit Stress on Germination and Early Seedling Growth

141

germination was closely related to the RL and in
severe stress condition the highest value of RL
was allocated to the drought-resistant line no. 9
(7219–69). Absolute increases in root elongation

rate are strongly related to a high water status in
plant organs.

Here we should distinguish between seed germi-

nation – which is completed when the radical

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

7112

191

181

436mo

7233-107 7233-12 BPKaraj

436-I

7219-69

Lines

Relative germination %

0
0.2
0.3

0

20

40

60

80

100

7112

191

181

436mo

7233-107 7233-12 BPKaraj

436-I

7219-69

Lines

Relative growth % CFW

0

20

40

60

80

100

7112

191

181

436mo

7233-1077233-12 BPKaraj

436-I

7219-69

Lines

Relative growth % RL

0
0.2
0.3

0

20

40

60

80

100

7112

191

181

436mo

7233-107 7233-12 BPKaraj

436-I

7219-69

Lines

Relative growth % RFW

0
0.2
0.3

0
0.2
0.3

(a)

(b)

(c)

(d)

Fig. 2: Seedlings of nine sugar beet lines compared at 0.0, 0.2, and 0.3 m concentrations of mannitol: (a) for the
relative germination %, (b) relative growth % of cotyledon fresh weight (CFW), (c) relative growth % of root length
(RL), and (d) relative growth % of root fresh weight (RFW). Error bars represent standard error

Table 3: Coefficients of correlation estimated between germination rate and in vitro seedling characteristics of sugar
beet lines

CDW

CFW

Ger.

in vitro

Ger.

in paper

Ger.

in perlit

Abnor.

RDW

RL

RFW

CDW

1

–

–

–

–

–

–

–

–

CFW

0.387

***

1

–

–

–

–

–

–

–

Ger. in vitro

0.512

***

0.648

***

1

–

–

–

–

–

–

Ger. in paper

0.347

***

0.214

0.42

***

1

–

–

–

–

–

Ger. in perlite

0.569

***

0.645

***

0.676

***

0.532

***

1

–

–

–

–

Abnor.

)0.466

***

)0.477

***

)0.685

***

)0.498

***

)0.665

***

1

–

–

–

RDW

0.273

*

0.325

**

0.449

***

0.177

0.332

*

)0.392

***

1

–

–

RL

0.553

***

0.715

***

0.698

***

0.275

*

0.750

***

)0.620

***

0.326

*

1

–

RFW

0.502

***

0.766

***

0.800

***

0.379

**

0.801

***

)0.621

***

0.403

**

0.799

***

1

Ger., germination %; Abnor., abnormality %; CFW, cotyledon fresh weight; CDW, cotyledon dry weight; RFW,
root fresh weight; RDW, root dry weight; RL, root length.
*P

¼ 0.05, **P ¼ 0.01, ***P ¼ 0.001.

142

Sadeghian and Yavari

expands and penetrates the medium, consisting of
only cell elongation – and the cell division and
radical growth (RL), which starts later on. Experi-
ments performed on Brassica oleracea var. italica
seeds in water stress medium demonstrated that the
sensitivity of radical expansion and radical growth
to water stress is markedly different (Bewley and
Black 1985). While germination % of these seeds
decline as water stress increase from 0 to

)8 bar

and finally stopped at

)14 bar, radical growth

started at

)8 bar water potential, declined at )16

bar and stopped at

)22 bar.

Our preliminary test of sugar beet germination

using high mannitol concentrations up to

)12 bar

(data not shown) indicated that the stress levels
exceeding

)8 bar did not permit seed imbibition

and distinction among the seed lines. Therefore,
experimental mannitol concentrations (0.2 and
0.3 m) were chosen according to a better distribu-
tion of stress effects observed among the seed lines.
The most tolerant lines were to be discriminated for
their better performance at both levels of water
deficiency.

These results are in accordance with the results of

field evaluation trials (Sadeghian et al. 2000). These
findings agreed fairly with those reported for
legumes in which drought-tolerant lines could be
screened during seedling stages using nutrient
solutions containing mannitol (Grezesiak et al.
1996).

Distinction of significant differences in sugar beet

seedling growth and physiological performance in
water restriction stress lead to the conclusion that
these parameters, specifically germination rate and
seedling RL, could be used as criteria in screening
the most tolerant progeny lines against abiotic
stresses. In vitro controlled conditions seem to be
more amenable for evaluation of genetic materials
at early growth phase. Progeny lines having stable
germination and seedling growth properties against
a range of induced osmotic pressures may then be
included in breeding programmes for yield poten-
tial and stability under water-restricted conditions
in experimental plots.

Acknowledgements

Financial assistance by Agricultural Research and Educa-
tion Organisation (AREO) and the assistance extended by
Seed Laboratory Technology and Tissue Culture Laborat-
ory of the Sugar Beet Seed Institute (SBSI) are gratefully
acknowledged. The authors also thank H. Ghaffari for his
critical comments on the manuscript.

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