J. Agronomy & Crop Science 190, 138 144 (2004)
Ó 2004 Blackwell Verlag, Berlin
ISSN 0931-2250
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 full growing period of other economically import-
ant crops such as wheat. Each year the sugar beet
Sugar beet progeny lines screened for both high water use
efficiency and high sugar yield under drought stress seedlings suffer due to lack or delay of irrigation
conditions in the field were assessed for the rate of seed
that hampers plant growth, affects final plant
germination and early seedling growth in water deficit
density and ultimately the root yield of the crop.
stress, induced by mannitol solutions. Seeds of nine
Seed germination is the most important seed
different sugar beet progeny lines were grown in three
quality, and germination under laboratory condi-
experimental conditions using filter paper, perlite and water
tions and field establishment are closely related
agar as substrate. Three levels of 0.0, 0.2 and 0.3 m
characteristics that are influenced by genetic (vari-
mannitol concentrations were applied in each experiment.
etal), environmental (location and year) and seed-
A factorial design was used with three replications.
Germination percentage was determined in all experiments. processing (treatments) effects (Apostolides and
Seedling growth parameters such as cotyledon fresh weight,
Goulas 1998). Studies on abiotic stress tolerance in
cotyledon dry weight, root fresh weight, root dry weight
sugar beet have been undertaken for the identifi-
(RDW) and root length (RL) were measured experiment-
cation of physiological and environmental factors.
ally. Abnormality was only recorded in the filter paper
Germination percentage of sugar beet measured
experiment. The results showed that drought stress could
under standard conditions correlated with the
be simulated by mannitol solution and significant differ-
seedling establishment in the field under stress
ences were found between stress levels for seedling charac-
conditions (Durrant et al. 1985, Durrant and
teristics. Distinct genetic variances were found among
progeny lines with respect to germination and early seedling
Gummerson 1990). Unfavourable germination
growth characteristics, except for cotyledons and RDW.
conditions such as low temperature and water
Seedling growth and germination rates severely declined at
stress have serious impacts on the results of vigour
the highest concentration of mannitol. The rate of abnor-
tests and plant establishment of diploid and trip-
mality was increased progressively at the germination stage
loid sugar beets in the field (Van Swaaij et al. 2001).
with an increase in mannitol concentration but it was more
Delayed seedling emergence of sugar beet was
pronounced in the drought-susceptible progeny lines. The
found to occur as a result of water stress and
highest values of relative germination % and relative
growth % of RL were obtained for the most tolerant line. increasing salinity (Ayers 1952). The effects of
In conclusion, seedling characteristics, in addition to other
abiotic stresses were studied on young sugar beet
physiological components involved in the seed germination
seedlings in the laboratory using osmotic agents
process under specific stress conditions, may be considered
such as mannitol, polyethylene glycol (PEG) and
for breeding purposes.
salts in Petri dishes containing Whatman filter
Key words: germination  in vitro  line-seedling- paper (Ghoulam and Fares 2001).
characteristics  mannitol  stress  sugar beet Small differences in the concentration of NaCl
did not change number of germinated seeds but
greatly affected water uptake and seedling growth
Introduction
(Durrant et al. 1974). Kaffka and Hembree (1999),
Spring-sown sugar beet crop in semi-arid condi- investigating the effect of seed priming on emer-
tions needs to be irrigated. This coincides with the gence rate and seedling growth of sugar beet in
U.S. Copyright Clearance Centre Code Statement: 0931 2250/2004/9002 0138 $15.00/0 www.blackwell-synergy.com
Effect of Water-Deficit Stress on Germination and Early Seedling Growth 139
saline soils, reported a similar rate of germination components. As several of these components are
for primed seeds in saline plots and non-primed difficult to measure, indirect selection should be
seeds in plots with low electric conductivity of soil applied (Visser 1994). Screening for drought toler-
(ECe). This practice is certain to become a seed- ance has been reported in vegetable crops using
enhancing requirement for crop production under mannitol as the stress agent (Grezesiak et al. 1996).
stressed conditions. Simulated drought condition with mannitol solu-
Nevertheless, breeders assume that there is con- tion was used to estimate drought resistance in
siderable variability in abiotic stress tolerance in 18 cultivars of field been (Vicia faba), soya bean
sugar beet germplasm. Hanson and Wyse (1982) (Glycine max), field pea (Pisum sativum), and lupin
evaluated sugar beet, fodder beet and Beta mari- (Lupinus albus). The results support the varietal
tima under increasing salinity conditions, and differences in seed germination and seedling growth
concluded that salinization increased betaine levels parameters. Osmotic adjustment in tolerant plant
of roots and shoots two- to three-fold. Beta helps maintain leaf metabolism and root growth at
maritima accessions accumulated 40 % less betaine relatively low leaf water potentials by controlling
in shoots than other progeny lines. Studies on turgor pressure in the cells. The trait can be assayed
abiotic stress tolerance in sugar beet have been easily by measuring growth rate of seedlings in
undertaken for the identification of physiological PEG solution (Morgan and Condon 1986). More-
and environmental factors. Productivity and ger- over, tolerance ratings under laboratory conditions
mination are linked to key properties of varieties were consistent with ratings of the effects of soil
used by beet growers and tend to decide the drought on yield in the field experiments, suggest-
ultimate crop yield (Tugnoli and Bettini 2001). ing that nutrient solution containing mannitol
Significant variations were found for total dry could be used in screening for drought tolerance
matter, root yield, sugar yield, juice purity and in growing seedlings.
impurities of roots among sugar beet, fodder beet Water deficit is one of the main limiting factors
and red beet when exposed to various periods of of sugar beet production in arid and semi-arid
drought stresses (Sadeghian et al. 2000). lands, therefore breeding programmes should be
The possibility of growing plants in controlled explicitly directed towards development of
conditions of greenhouse and laboratory are of drought-tolerant varieties. We report the effects of
great convenience for the evaluation of genetic an increasing stress agent (mannitol concentra-
traits. In many crops, seed germination and early tions) on the germination and seedling growth in
seedling growth are the stages most sensitive sugar beet lines, with an aim to choose an
to environmental stresses (Cook 1979). Foolad evaluation procedure for the identification of sugar
and Lin (1999) studied the germination response beet tolerant to water stress at germination and
of tomato accessions in Petri dishes containing early growth phase.
agar media treated either with 0 mm NaCl (non-
stress and cold-stress treatments) or 150 mm
Materials and Methods
NaCl + 15 mm CaCl2 (for salt-stress treatment).
Nine sugar beet progeny lines  one very drought tolerant,
They concluded that some of the same genetic and
four tolerant, two moderate, one rather sensitive and one
physiological parameters contributing to rapid seed
sensitive (S1 and S2 families)  were previously identified
germination under non-stress conditions could also
(Sadeghian et al. 2000) under field condition for their good
facilitate rapid seed germination under stress.
yield performances under drought conditions (Table 1).
Experiments performed at germination and seed-
Seeds of these progeny lines were produced in the same seed
ling emergence stages of sugar beet under various
production region, and were graded, standardized, washed,
temperatures and NaCl concentrations indicated
sterilized and dried at room temperature prior to the
that the effect of salinity on seedling emergence was experiments. Germination tests were conducted in three
experiments consisting of filter paper (ISTA standard seed
increasingly inhibitory as temperature increased
testing conditions and at 22 Ä… 2 °C), box containing
from 10 15 °C to 25 and 35 °C (Mahmoud and
perlite (greenhouse conditions with night/day temperature
Hill 1981). Small differences in the concentration of
fluctuations from 15 to 23 °C), and 0.8 % water-agar
NaCl did not change the rate of germinated seeds
medium in plates (in vitro growth conditions at 25 Ä… 2 °C).
but greatly affected water uptake and seedling
Stress treatments were performed at 0.0 (control), 0.2 and
growth (Durrant et al. 1974).
0.3 m concentrations of mannitol approximately corres-
The common approach in breeding for drought
ponding to )1, )5 and )7 bar osmotic pressures. In the
greenhouse experiment, boxes (37 · 55 · 13 cm) were
tolerance of crops is to select for drought tolerance
140 Sadeghian and Yavari
Table 1: Brief descriptions of sugar beet lines evaluated All measured variables were subjected to analysis of
in mannitol-stressed conditions variance (anova) and SAS software was used for the
correlation analysis (SAS Institute, Inc. 1996). Sugar beet
Drought
lines were compared for germination %, abnormal seedling
Line no. Identity no. Seed type tolerance
% and relative growth % of seedling characteristics.
1 7112 mm S
2 191 MM RS
Results and Discussion
3 181 mm MT
4 436m MM MT
Records were made on the number of germinated
5 7233-17 MM T
seed in paper, perlite, and agar medium and of
6 7233-12 MM T
seedling growth characteristics under in vitro con-
7 BP-Karadj MM T
dition. Analysis of variance showed significant
8 463-I-BMM T
effects of mannitol treatments and progeny lines
9 7219-69 MM VT
(P < 0.001) on the rate of germination in the three
MM, multigerm; mm, monogerm; S, sensitive; RS, rather experiments (Table 2). Germination was progres-
sensitive; MT, medium tolerant; VT, very tolerant.
sively inhibited by the increase in mannitol
concentration. The strongest inhibition occurred
watered with 1/2 diluted Hoagland solution (Hoagland and
at the second mannitol concentration (0.3 m) par-
Arnon 1950) for each different mannitol level. The amount
ticularly in perlite and water agar medium. The
of this solution was adjusted daily to keep a 2-cm level at
response of seeds to an increase in mannitol con-
the bottom. A factorial design with three replications was
centration varied among progeny lines (Fig. 1a c).
used for each experiment and each treatment replicate
Drought stress simulated by mannitol and its
contained 100 seeds/box except in the agar medium, where
25 seeds were grown individually in culture tubes. effects on all seedling characteristics were signifi-
Germination was recorded in all the three experiments,
cant (Table 2). The seedling growth parameters
and percentage of abnormal seedlings (germinated seeds
were significantly affected by an increase in the
failed in growing standard roots and shoots) was also
osmotic pressure of mannitol concentrations
calculated in the filter paper test. Germination percentage
(Fig. 2b d). Except for very small values obtained
was recorded after 14 days in paper, 24 days in perlite and
for CDW and RDW, significant differences among
28 days in agar medium. Seedling characteristics such as
the progeny lines (P < 0.01) were obtained for the
cotyledon fresh weight (CFW), cotyledon dry weight
seedling growth traits (Table 2). The effect of
(CDW), root fresh weight (RFW), root dry weight
(RDW) and root length (RL) were measured in the water decreasing water potential in 0.2 and 0.3 m mann-
agar medium. Assessments of seedling characteristics were
itol concentrations resulted in seedlings with less
made 28 days after sowing. The relative germination was
fresh weight during the processes of germination
determined by the following calculation: number of germi-
and early growth when compared with the control
nated seeds in stress medium/number of germinated seeds
treatment (Figs 1 and 2). Analysis of variance for
in control medium · 100 (Smith and Dobrenz 1987).
CFW and RFW indicated that stress condition and
Similarly, relative growth % for RL, RFW and CFW
progeny lines had significant differences for these
was calculated for various progeny lines in the in vitro
parameters. Stresses decreased water content and
experiment.
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 Ger. Ger. Ger.
variation d.f. in paper in perlite 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 · 16 134.8827*** 230.734*** 399.964*** 0.01356 0.0002 0.0005 0.00005 43.217
mannitol
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 ź 0.01, ***P ź 0.001.
Effect of Water-Deficit Stress on Germination and Early Seedling Growth 141
(a) (b)100 0
0
100
0.2 90
0.2
90
80
80
0.3
0.3
70
70
60
60
50
50
40
40
30 30
20
20
10 10
0 0
Lines Lines
90
(c) 0 (d)
100
0
80
90
0.2
0.2
70
80
0.3
0.3
70 60
60
50
50
40
40
30
30
20
20
10
10
0
0
Lines Lines
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
assimilate accumulation in the seedling as a conse- a decrease for all growth parameters as stress
quence of osmotic pressure induced by mannitol. levels intensified. The most tolerant seedlings
Interaction effects between genotype and mannitol showed a better biological efficiency (increase in
concentration were significant for the germination size and weight) under increased water deficiency,
rates in all cases, pointing to the fact that differ- while a different distribution of biomass in leaves
ences in genetic components affecting the germina- and roots were noted (Fig. 2b,d). Here again
tion response of sugar beet are expressed at early line no. 9 (7219 69) responded best for relative
stages under specific stress conditions. As shown in germination and relative growth of RL. Various
Fig. 1a, the germination percentage recorded in progeny lines responded differently for the relat-
paper was only slightly affected but number of ive growth of CFW and RFW (Fig. 2b d). A
abnormal seedlings augmented, as the stress was positive correlation was found between germina-
intensified (Fig. 1d). The differences in germination tion rates and seedling characteristics in the three
percentage of seeds subjected to stress levels were experiments, except for the percentage of abnor-
more detectable in the perlite and water agar mal seedling (Table 3). Coefficients of correlation
experiments (Fig. 1b,c). Germination of seed in line between seed germination in paper with that of
nos 2 (191), 1 (7112) and 3 (181) proved to be most perlite and agar medium were strongly significant
sensitive to water restriction at the higher level of (P < 0.001). Abnormality always had a negative
osmotic potential in the in vitro experiment, but correlation with germination rates and other
seeds of line no. 9 (7219) revealed a relatively stable seedling characteristics like CDW, CFW, RDW,
tolerance in both stress levels (Fig. 1c). RL and RFW, indicating that seedling water
Evaluation of seedling characteristics presented potential and growth efficiency have an essential
as a percentage of the control (Fig. 2a d) showed role in the development of normal plants. Seed
GER. % in Paper
GER. % in perlite
Abnor. %
GER. % in Vitro
191
181
191
181
7112
7112
436-I
436-I
436mo
436mo
7233-12
BPKaraj
7219-69
7233-12
BPKaraj
7219-69
7233-107
7233-107
191
181
191
181
7112
7112
436-I
436-I
436mo
436mo
7233-12
BPKaraj
7219-69
7233-12
BPKaraj
7219-69
7233-107
7233-107
142 Sadeghian and Yavari
(a) (b)
0
0
0.2
0.2
0.3
0.7
0.3
100
0.6
80
0.5
0.4
60
0.3
40
0.2
20
0.1
0 0
Lines Lines
(c) (d)
0
0
0.2
0.2
0.3
0.3
100
100
80
80
60
60
40
40
20 20
0 0
Lines Lines
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
Ger. Ger. Ger.
CDW CFW in vitro in paper 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 ź 0.05, **P ź 0.01, ***P ź 0.001.
germination was closely related to the RL and in rate are strongly related to a high water status in
severe stress condition the highest value of RL plant organs.
was allocated to the drought-resistant line no. 9 Here we should distinguish between seed germi-
(7219 69). Absolute increases in root elongation nation  which is completed when the radical
Relative germination %
Relative growth % CFW
Relative growth % RL
Relative growth % RFW
191
181
191
181
7112
7112
436-I
436-I
436mo
436mo
7233-12
BPKaraj
7219-69
7233-12
BPKaraj
7219-69
7233-107
7233-107
191
181
191
181
7112
7112
436-I
436-I
436mo
436mo
BPKaraj
7233-12
7219-69
7233-12
BPKaraj
7219-69
7233-107
7233-107
Effect of Water-Deficit Stress on Germination and Early Seedling Growth 143
expands and penetrates the medium, consisting of
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