J.

Agronomy & Crop Science (2013) ISSN 0931-2250

DROUGHT STRESS

Seed Priming with Ascorbic Acid Improves Drought

Resistance of Wheat
M. Farooq1,2, M. Irfan1, T. Aziz1, I. Ahmad1 & S. A. Cheema1
1 Department of Agronomy, University of Agriculture, Faisalabad, Pakistan
2 The UWA Institute of Agriculture, The University of Western Australia, Crawley, WA, Australia

Keywords Abstract
ascorbate; drought; osmotic adjustment; seed
priming; water relations The study, consisting of two independent experiments, was conducted to evalu-
ate the role of seed priming with ascorbic acid (AsA) in drought resistance of
Correspondence wheat. In the first experiment, seeds of wheat cultivars Mairaj-2008 and Lasani-
M. Farooq 2008 were either soaked in aerated water (hydropriming) for 10 h or not
Department of Agronomy
soaked (control). In the second experiment, seeds of same wheat cultivars were
University of Agriculture
Faisalabad-38040
soaked in aerated (2 mm) AsA solution (osmopriming) or water (hydropri-
Pakistan ming) for 10 h. In both experiments, seeds were sown in plastic pots (10 kg)
Tel.: +92 41 9200161 9/2931 maintained at 70 % and 35 % of water-holding capacity designated as well
Fax: +92 41 9200605 watered and drought stressed, respectively. Both experiments were laid out in a
Email: farooqcp@gmail.com; completely randomized design with six replications. Drought caused delayed
mfarooq@uaf.edu.pk and erratic emergence and disturbed the plant water relations, chlorophyll con-
tents and membranes because of oxidative damage; however, root length in
Accepted April 20, 2012
cultivar Lasani-2008 was increased under drought. Hydropriming significantly
doi:10.1111/j.1439-037X.2012.00521.x improved the seedling emergence and early growth under drought and well-
watered conditions; however, improvement was substantially higher from
osmopriming with AsA. Similarly, osmopriming with AsA significantly
improved the leaf emergence and elongation, leaf area, specific leaf area, chlo-
rophyll contents, root length and seedling dry weight. Owing to increase in
proline accumulation, phenolics and AsA, by seed priming with AsA, plant
water status was improved with simultaneous decrease in oxidative damages.
These improved the leaf emergence and elongation, and shoot and root growth
under drought. However, there was no difference between the cultivars in this
regard. In conclusion, osmopriming with AsA improved the drought resistance
of wheat owing to proline accumulation and antioxidant action of AsA and
phenolics, leading to tissue water maintenance, membrane stability, and better
and uniform seedling stand and growth.

Water deficit during initial stage of crop results in

Introduction
Wheat (Triticum aestivum L.), the most important winter
cereal, is the staple for millions around the world.
Drought is the major factor limiting crop growth and
productivity in many regions of the world, the loss of
which is more than any other single environmental factor
(Farooq et al. 2009a,b). However, changing global climate
is making the situation more serious (IPCC 2007).
delayed and erratic seedling emergence and stand establish-
ment (Almansouri et al. 2001, Kaya et al. 2006), and in
severe cases, complete inhibition of seedling emergence
may also result (Kaya et al. 2006). Decrease in water uptake
during imbibition phase of germination is the primary rea-
son for this decline in stand establishment (Muril-
lo-Amador et al. 2002). Drought also disturbs the plant
growth owing to loss of turgor (Farooq et al. 2009a, Taiz

12 ª 2012 Blackwell Verlag GmbH, 199 (2013) 12–22


and Zeiger 2010), as water supply from the xylem to the
surrounding elongating cells is interrupted (Nonami 1998).
Production and accumulation of osmoprotectants, for
example, proline, glycinebetaine in plant tissues during
drought, the osmotic adjustment, is an adaptive response
(Vendruscolo et al. 2007, Cattivelli et al. 2008, Farooq
et al. 2008a, Izanloo et al. 2008, Hussain et al. 2009).
Osmotic adjustment helps in improving water uptake
(Tangpremsri et al. 1991, Chimenti et al. 2006, Farooq
et al. 2009a) and enables leaf turgor maintenance for the
same leaf water potential, thus supporting stomatal con-
ductance and carbon assimilation under drought (Ali
et al. 1999, Farooq et al. 2009a).
Over-production of reactive oxygen species (ROS) than
their dousing is one of the key responses of plants to
environmental stresses (Smirnoff 1998, Farooq et al.
2009a, 2011). The ROS, thus generated, deteriorate the
cellular membranes and several other vital substances and
may even lead to cell death (Beligni and Lamattina 1999,
Kratsch and Wise 2000). Malondialdehyde (MDA) pro-
duction is taken as an index of ROS-induced oxidative
damage (Teisseire and Guy 2000, Zhang et al. 2007).
However, plants have evolved several antioxidative
defence mechanisms, including the production of enzy-
matic and non-enzymatic antioxidants to reduce ROS-
induced oxidative damages (Posmyk et al. 2009).
Seed priming, a controlled hydration technique that
allows the pre-germination metabolisms without actual
germination (Bradford 1986, Farooq et al. 2009c), is one
of the most pragmatic and short-term approaches to
combat the effects of drought (Kaya et al. 2006, Farooq
et al. 2010) and other environmental stresses (Farooq
et al. 2008b,c, 2010, Jafar et al. 2012) on seedling emer-
gence and stand establishment. Primed seeds usually have
higher and synchronized germination (Brocklehurst et al.
1984, Kaya et al. 2006, Farooq et al. 2009c) owing to sim-
ply a reduction in the lag time of imbibitions (Brockle-
hurst and Dearman 2008), build-up of germination-
enhancing metabolites (Farooq et al. 2006a), metabolic
repair during imbibition (Burgass and Powell 1984, Bray
et al. 1989) and osmotic adjustment (Bradford 1986).
Ascorbic acid (AsA) is one of the important metabo-
lites involved in cell division, osmotic adjustment
(De-Gara et al. 2003) and also plays vital role during the
initial stages of germination (Arrigoni et al. 1997). Ascor-
bic acid also possesses strong antioxidant potential and
helps in balancing the production and scavenging of ROS
(Müller-Moulé et al. 2003, 2004); however; high endoge-
nous AsA level is required to maintain the balance. Inter-
estingly, exogenous application of AsA can increase the
endogenous AsA level (Chen and Gallie 2004).
Application of AsA through seed priming may thus be
helpful in improving the stand establishment and allome-
ª 2012 Blackwell Verlag GmbH, 199 (2013) 12–22
Ascorbic Acid Improves Drought Resistance of Wheat
try of wheat under drought. This study was conducted to
evaluate the potential of AsA in improving the drought
resistance in wheat. It was hypothesized that seed priming
with AsA improves the drought resistance of wheat
through increase in endogenous AsA contents,
antioxidant potential and osmotic adjustment.
Materials and Methods
Plant material
Seeds of wheat cultivars ‘Lasani-2008’ and ‘Mairaj-2008’,
used in this study, were obtained from Wheat Research
Institute, Faisalabad, Pakistan and Regional Agriculture
Research Institute, Bahawalpur, Pakistan, respectively. Ini-
tial moisture contents and germination percentage were
9.14 %, 9.04 % and 95.5 %, 96.25 % in cultivars Mairaj-
2008 and Lasani-2008, respectively.
Experimental details
The study consisted of two independent experiments. In
the first experiment, seeds of both wheat cultivars were
either soaked in aerated water (hydropriming) for 10 h or
not soaked (control). In the second experiment, wheat
seeds of both cultivars were soaked in aerated 2 mm solu-
tion of ascorbic acid (AsA; osmopriming) or distilled
water (hydropriming) for 10 h, keeping seed to solution
ratio of 1 : 5 (w/v) (Farooq et al. 2006b). Seeds were then
removed, rinsed thoroughly with distilled water and
re-dried near to their original weight with forced air at
27 C ± 2 under shade.
In both experiments, seeds were sown (eight in each
pot) in soil-filled (10 kg) plastic pots maintained at 70 %
and 35 % of water-holding capacity designated as well
watered and drought stressed, respectively. Plants were
thinned to four plants per pot after achieving the con-
stant count. Soil moisture was monitored and maintained
every alternate day. Experimental soil was sandy loam
having ECe 1.65 dS m)1 and pH 7.8. Both experiments
were laid out in a completely randomized design in a
factorial arrangement with six replicates per treatment.
Stand establishment
Seedling emergence was observed daily according to the
Association of Official Seed Analysts (AOSA) (1990) until
a constant count was achieved. The time to 50 % emer-
gence (E50) was calculated following the method of Farooq
et al. (2005). Mean emergence time (MET) was calculated
according to the equation of Ellis and Roberts (1981).
Coefficient of uniformity of emergence (CUE) was calcu-
lated using the formulae of Bewley and Black (1994).
13
Farooq et al.
Allometry
Number of leaves was counted daily, starting from the
seedling emergence; length of each individual leaf was
measured daily to derive the leaf elongation rates. Four
weeks after the emergence, plants were harvested to
record root and shoot lengths and seedling dry weight.
Leaf area was measured manually with a ruler while spe-
cific leaf area (SLA) was calculated as the ratio of leaf area
to leaf weight.
Plant water relations
Leaf water potential (ww) of penultimate leaves was mea-
sured by pressure chamber (Model 3005; Soil Moisture
Equipment Corp., Santa Barbara, CA, USA) between
6 : 00 and 9 : 00 am 1 day before the final harvest. The
same leaves were put in a glass vial and stored at )20 C
for 24 h. After thawing at room temperature (for
15 min), cell sap was extracted, and the osmotic potential
(ws) was measured using vapour pressure osmometer
(Model 5500; Wescor Inc., Logan, UT, USA). The relative
leaf water contents (RWC) were measured following the
technique of Barrs and Weatherly (1962).
Photosynthetic pigments
Photosynthetic pigments, chlorophyll-a and -b, were
determined following Arnon (1949) using 500 mg fresh
leaf extracted overnight with 80 % acetone and centri-
fuged at 14 000 g for 5 min.
Membrane stability index
Membrane stability was estimated by measuring the con-
ductivity of leachates owing to damaged plasma mem-
brane following the method of Shanahan et al. (1990).
One gram of leaf material (10 · 10 mm pieces) was taken
in 10 ml distilled water in glass vials and kept at 10 C
for 24 h with shaking. The initial conductivity (C1) was
recorded after bringing sample to 25 C with conductivity
meter. The samples were then autoclaved for 10 min,
cooled to 25 C, and final conductivity (C2) was
recorded. Membrane stability index (MSI) was calculated
as MSI = [1 ) (C1/C2)] · 100.
Metabolite determination
Oxidative damage to the membrane lipids was estimated
by analysing the content of total thiobarbituric acid–reac-
tive substances (TBARS), expressed as equivalents of mal-
ondialdehyde (MDA). The amount of MDA was
determined following Hichem et al. (2009). For the
14
estimation of total phenolics, leaf samples were extracted
with 95 % methanol, and the phenolics were estimated
using Folin–Ciocalteu method (Ainsworth and Gillespie
2007). To determine the amount of free proline, fresh leaf
material was homogenized in 3 % aqueous sulfosalicylic
acid, and free leaf proline was estimated following the
method of Bates et al. (1973). To determine ascorbic acid,
leaves were homogenized in 6 % trichloroacetic acid and
ascorbic acid was estimated according to the procedure of
Mukherjee and Choudhuri (1983).
Statistical analysis
Data collected were subjected to statistical analysis by
analysis of variance using the computer software costat
(Cohort Software, Berkeley, CA, USA). The mean values
were compared with the least significance difference test
following the procedure of Snedecor and Cochran (1980).
Microsoft Excel was used for the graphical presentation
and calculation of correlation coefficients.
Results
Experiment 1
Drought significantly delayed the MET and E50; however,
hydropriming significantly decreased the MET and E50
under drought and well-watered conditions (Table 1).
Likewise, drought decreased the CUE and seedling dry
weight in both cultivars, hydropriming but improved
CUE and seedling dry weight under drought and well-
watered conditions (Table 1).
Experiment 2
In both wheat cultivars, MET, E50 and CUE were signifi-
cantly affected by drought and seed priming (Table 2).
Drought increased the MET and E50 in wheat cultivars;
however, osmopriming with AsA significantly decreased
the MET and E50 both under drought and well-watered
conditions than hydropriming (Table 2). Likewise
drought decreased the CUE; nonetheless, osmopriming
with AsA substantially improved that under control and
drought conditions than hydropriming (Table 2).
Drought stress significantly decreased the seedling dry
weight (Table 1), shoot length, leaf area and specific leaf
area (SLA) (Table 3) in both wheat cultivars. However,
osmopriming with AsA significantly improved seedling
dry weight (Table 3), shoot length, leaf area and SLA in
wheat cultivars Lasani-2008 and Mairaj-2008 (Table 3).
Although root length was increased under drought in cul-
tivar Lasani-2008, there was no difference for root length
in cultivar Mairaj-2008 under drought and well-watered
ª 2012 Blackwell Verlag GmbH, 199 (2013) 12–22
 Ascorbic Acid Improves Drought Resistance of Wheat

Table 1 Influence of hydropriming on stand establishment and seedling dry weight in wheat cultivars under well-watered and drought conditions

MET (days) E50 (days) CUE Seedling dry weight (g)

Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008

Treatments WW DS WW DS WW DS WW DS WW DS WW DS WW DS WW DS

Control 8.44c 9.65a 8.23d 9.09b 6.33e 8.18a 6.52d 8.00b 0.45c 0.29e 0.42c 0.28e 0.13b 0.09d 0.14b 0.09d
Hydropriming 7.41e 8.33c,d 7.49e 8.43c 5.71f 7.19c 5.87f 7.11c 0.71a 0.37d 0.68b 0.36d 0.17a 0.11c 0.18a 0.12b,c

Means sharing the same letter for a single parameter do not differ significantly at P < 0.05.
WW, well-watered; DS, drought stress; MET, mean emergence time; E50, time to 50 % emergence; CUE, coefficient of uniformity of emergence.

Table 2 Influence of seed priming with ascorbic acid on stand establishment and seedling dry weight in wheat cultivars under well-watered and
drought conditions

MET (days) E50 (days) CUE Seedling dry weight (g)

Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008

Treatments WW DS WW DS WW DS WW DS WW DS WW DS WW DS WW DS

Hydropriming 7.29b 8.09a 7.23b 8.09a 5.66e 7.08a 5.92d,e 7.00a,b 0.67b 0.33d 0.62b 0.31d 0.151d 0.113g 0.161c 0.117f
Osmopriming 6.47c 7.42b 6.29c 7.25b 4.80f 6.20c,d 5.04f 6.53b,c 0.82a 0.41c 0.78a 0.43c 0.169b 0.123e 0.178a 0.126e

Means sharing the same letter for a single parameter do not differ significantly at P < 0.05.
WW, well-watered; DS, drought stress; MET, mean emergence time; E50, time to 50 % emergence; CUE, coefficient of uniformity of emergence.

Table 3 Influence of seed priming with ascorbic acid on root and shoot lengths, leaf area and specific leaf area in wheat cultivars under well-
watered and drought conditions

Root length (cm) Shoot length (cm) Leaf area (cm2) Specific leaf area (cm2 g)1)

Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008

Treatments WW DS WW DS WW DS WW DS WW DS WW DS WW DS WW DS

Hydropriming 8.82e 9.79b,c 9.63c,d 9.13d,e 5.29b,c 3.67e 5.39b 3.92e 15.77d 13.80e 17.95c 15.65d 257.22b 253.71d 256.76b 253.89d
Osmopriming 9.79b,c 10.55a 10.07a,b,c 10.20a,b 5.47a,b 4.50d 5.90a 4.92cd 19.87b 17.47c 21.37a 20.33b 259.16a 255.31c 259.78a 255.55c

Means sharing the same letter for a single parameter do not differ significantly at P < 0.05.
WW, well-watered; DS, drought stress.

conditions (Table 3). However, in all cases, osmopriming
with AsA substantially increased the root length (Table 3).
Maximum root length was recorded from cultivar Lasani-
2008 under drought raised from seeds osmoprimed with
AsA followed by cultivar Mairaj-2008 under same
conditions (Table 3).
Drought substantially delayed the leaf emergence and
decreased the number of leaves (Fig. 1) and elongation of
1st (Fig. 2a), 2nd (Fig. 2b), 3rd (Fig. 2c) and 4th
(Fig. 2d) leaf in both wheat cultivars. Nonetheless, seed
priming with AsA substantially improved the leaf emer-
gence (Fig. 1) and elongation of 2nd (Fig. 2b), 3rd
(Fig. 2c) and 4th (Fig. 2d) leaf under well-watered and
drought conditions in both wheat cultivars. However, the
elongation of 1st leaf was improved by seed priming with
AsA under well-watered conditions in both cultivars,
whereas under drought, seed priming with AsA improved
the elongation of 1st leaf in cultivar Mairaj-2008 only
(Fig. 2a).
Drought significantly decreased the leaf water potential,
osmotic potential and relative leaf water contents in both
cultivars (Table 4). Although, there was no difference in
leaf water potential from hydropriming and osmopriming
with AsA under well-watered conditions in cultivar
Lasani-2008 and under both well-watered and drought
conditions in cultivar Mairaj-2008, leaf water potential
was higher in cultivar Lasani-2008 osmoprimed with AsA
under drought than hydroprimed seeds (Table 4). There
was no statistical difference between hydropriming and
osmopriming with AsA under well-watered conditions in
cultivar Lasani-2008 for osmotic potential; however, in
rest of the cases, osmotic potential was higher from

ª 2012 Blackwell Verlag GmbH, 199 (2013) 12–22 15

Farooq et al.

5 (a) Osmopriming
Hydropriming
4

1
Number of leaves per plant

0
10 12 14 16 18 20 22 24 26 11 13 15 17 19 21 23 25

5 (b) Osmopriming
Hydropriming

0
10 12 14 16 18 20 22 24 26 11 13 15 17 19 21 23 25 Fig. 1 Influence of seed priming with ascor-
bic acid on leaf emergence in wheat cultivar
Well-watered Drought (a) Lasani-2008 and (b) Mairaj-2008 under
Days after sowing well-watered and drought conditions ±S.E.

osmopriming than hydropriming (Table 4). Osmopriming
with AsA significantly improved the relative leaf water
contents in both cultivars under both well-watered and
drought conditions (Table 4).
Drought stress significantly decreased the chlorophyll
contents in both wheat cultivars. In both cultivars, chlo-
rophyll-a contents were substantially improved by osmo-
priming with AsA than hydropriming under both
drought and well-watered conditions. However, chloro-
phyll-b contents were improved by osmopriming with
AsA only under well-watered conditions but not under
drought (Table 5).
Drought significantly disturbed the membrane stability
in both cultivars (Table 5). Although there was no differ-
ence for membrane stability between hydropriming and
osmopriming with AsA under well-watered conditions in
both wheat cultivars, under drought, osmopriming with
AsA substantially increased the membrane stability in
both wheat cultivars (Table 5).
Malondialdehyde contents, an index of oxidative stress,
were substantially increased under drought in both wheat
cultivars (Table 6); however, osmopriming with AsA sub-
stantially decreased the MDA contents in both wheat cul-
tivars (Table 6). Likewise, soluble phenolics, leaf proline
contents and ascorbic acid contents were also increased
under drought in both cultivars (Table 6). Osmopriming
with AsA significantly increased soluble phenolics, leaf
proline contents and ascorbic acid contents in both culti-
vars under drought and well-watered conditions
(Table 6). There was no difference between both cultivars
for MDA, soluble phenolics and leaf proline contents
under drought and well-watered condition; however,
under well-watered conditions, ascorbic acid contents
were higher in cultivar Lasani-2008 while under drought,
ascorbic acid contents were higher in cultivar Mairaj-2008
(Table 6).
Under well-watered conditions, CUE was positively
correlated with SLA, chlorophyll-a and proline contents;

16 ª 2012 Blackwell Verlag GmbH, 199 (2013) 12–22

 Ascorbic Acid Improves Drought Resistance of Wheat

8 (a) 8 (a)

6 6

4 4

2 2
Osmopriming
Osmopriming
Hydropriming
Hydropriming
0 0
11 13 15 17 19 21 11 13 15 17 19 21 11 13 15 17 19 21 11 13 15 17 19 21

12 (b) 12 (b)
10 10

8 8

6 6

4 4
Osmopriming Osmopriming
2 2
Hydropriming
Hydropriming
Leaf length (cm)

0 0
14 16 18 20 22 24 26 28 15 17 19 21 23 25 27 14 16 18 20 22 24 26 28 15 17 19 21 23 25 27

14 Osmopriming 14 Osmopriming
(c) (c)
Hydropriming Hydropriming
12 12
10 10
8 8

6 6
4 4

2 2

0 0
15 17 19 21 23 25 27 15 17 19 21 23 25 27 15 17 19 21 23 25 27 16 18 20 22 24 26 28

8 (d) Osmopriming 8 (d) Osmopriming

Hydropriming Hydropriming

6 6

4 4

2 2

0 0
20 21 22 23 24 25 26 27 28 22 23 24 25 26 27 28 20 21 22 23 24 25 26 27 28 22 23 24 25 26 27 28
Well-watered Drought Well-watered Drought
Lasani-2008 Mairaj-2008
Days after sowing

Fig. 2 Influence of seed priming with ascorbic acid on leaf elongation of (a) first, (b) second, (c) third and (d) fourth leaf in wheat cultivars Lasan-
i-2008 and Mairaj-2008 under well-watered and drought conditions ±S.E.

seedling dry weight had positive correlation with leaf area, stability, phenolics and proline contents (Table 7). While
SLA, chlorophyll-a, membrane stability, phenolics and SLA and chlorophyll-a had positive correlation with each
proline contents, and leaf area was positively correlated other and with CUE, seedling dry weight, leaf area, RWC,
with seedling dry weight, SLA, chlorophyll-a, membrane membrane stability, phenolics and proline contents, RWC

ª 2012 Blackwell Verlag GmbH, 199 (2013) 12–22 17

Farooq et al.

Table 4 Effect of seed priming with ascorbic acid on plant water relations in wheat cultivars under well-watered and drought conditions

Water potential ()MPa) Osmotic potential ()MPa) Relative leaf water contents (%)

Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008

Treatments WW DS WW DS WW DS WW DS WW DS WW DS

Hydropriming 0.146c 0.180a 0.113d 0.143c 1.68c,d 1.84a 1.70c 1.83a 90.55b,c 86.86e,f 89.95c 86.62f
Osmopriming 0.134c 0.162b 0.110d 0.134c 1.66d 1.79b 1.65d 1.76b 91.41a 87.99d 91.95a 87.52d,e

Means sharing the same letter for a single parameter do not differ significantly at P < 0.05.
WW, well-watered; DS, drought stress.

Table 5 Effect of seed priming with ascorbic acid on chlorophyll contents and membrane stability index in wheat cultivars under well-watered
and drought conditions

Chl-a (mg g)1 FW) Chl-b (mg g)1 FW) Membrane stability index (%)

Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008

Treatments WW DS WW DS WW DS WW DS WW DS WW DS

Hydropriming 6.76b 3.95d 6.56b 4.00d 2.82c 2.08d 3.21b 2.22d 43.88a,b 33.61d 45.34a 37.28c,d
Osmopriming 7.88a 5.31c 8.17a 5.27c 3.87a 2.25d 3.67a 2.33d 46.50a 38.74b,c 48.89a 39.93b,c

Means sharing the same letter for a single parameter do not differ significantly at P < 0.05.
WW, well-watered; DS, drought stress.

Table 6 Effect of seed priming with ascorbic acid on chlorophyll contents, soluble phenolics and membrane stability index in wheat cultivars
under well-watered and drought conditions

Leaf MDA content Leaf free proline contents

(lmol g)1FW) Soluble phenolics (mg g)1 FW) (lmol g)1 FW) Ascorbic acid (mg g)1 FW)

Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008

Treatments WW DS WW DS WW DS WW DS WW DS WW DS WW DS WW DS

Hydropriming 15.56c 21.29a 15.37c 22.03a 33.74e 43.76b 34.94e 44.31b 7.87e 12.37c 8.23e 14.45c 16.75e 27.05c 14.21f 29.87b
Osmopriming 13.35d 17.56b 13.67d 17.24b 36.95d 45.89a 38.48c 46.52a 9.81d 17.56a 9.75d 18.25a 18.23d 29.33b 16.44e 31.17a

Means sharing the same letter for a single parameter do not differ significantly at P < 0.05.
WW, well-watered; DS, drought stress.

was positively correlated with membrane stability, pheno-
lics and proline contents (Table 7). Membrane stability
index was positively correlated with phenolics and proline
contents; whereas, phenolics were positively correlated
with proline contents (Table 7). Under both well-watered
and drought conditions, MDA had negative correlation
with CUE, seedling dry weight, leaf area, SLA, chl-a,
RWC, membrane stability, phenolics and proline contents
(Tables 7 and 8).
Under drought, CUE had positive correlation with
seedling dry weight, leaf area, SLA, chlorophyll-a, RWC,
phenolics and proline contents; seedling dry weight was
positively correlated with leaf area, SLA, chlorophyll-a,
membrane stability, AsA, phenolics and proline contents
(Table 8). Leaf area was positively correlated with SLA,
chl-a, AsA, membrane stability, phenolics and proline
contents whereas SLA had positive correlation with chlo-
rophyll-a, RWC, membrane stability, AsA, phenolics and
proline contents and chlorophyll-a was positively corre-
lated with RWC, membrane stability, phenolics and pro-
line contents (Table 8). RWC had positive correlation
with phenolics and proline contents whereas membrane
stability index was positively correlated with AsA, pheno-
lics and proline contents and AsA and phenolics were
positively correlated with proline contents (Table 8).
Discussion
This study investigated whether seed priming with AsA
can improve drought resistance in wheat. Drought caused

18 ª 2012 Blackwell Verlag GmbH, 199 (2013) 12–22

 Ascorbic Acid Improves Drought Resistance of Wheat

Table 7 Correlation coefficients of important traits in wheat genotypes under well-watered conditions (n = 6)

DW LA SLA Chl-a RWC MSI AsA Phen Pro MDA

CUE 0.71ns 0.73ns 0.94** 0.94** 0.91* 0.67ns 0.73ns 0.79ns 0.97** )0.99**
DW 0.99** 0.87** 0.88* 0.80ns 0.99** 0.44ns 0.99** 0.98** )0.95**
LA 0.87** 0.88* 0.79ns 0.97** 0.24ns 0.99** 0.93** )0.88**
SLA 0.99** 0.99** 0.87** 0.63ns 0.93** 0.94** )0.93**
Chla-a 0.98** 0.87** 0.63ns 0.93** 0.95** )0.95**
RWC 0.82* 0.66ns 0.87* 0.87** )0.87**
MSI 0.16ns 0.86* 0.98** )0.80 ns
AsA 0.32ns 0.56ns )0.65ns
Phen 0.94** 0.90*
Pro 0.99**

CUE, coefficient of uniformity of emergence; DW, seedling dry weight; LA, leaf area; SLA, specific leaf area; Chl-a, chlorophyl-a; RWC, relative
leaf water contents; MSI, membrane stability index; AsA, ascorbic acid; Phen, total soluble phenolics; Pro, leaf free prolines; MDA, malondialde-
hyde; ns, non-significant. *Significant at P < 0.05; **Significant at P < 0.01.

Table 8 Correlation coefficients of important traits in wheat genotypes under drought conditions (n = 6)

DW LA SLA Chl-a RWC MSI AsA Phen Pro MDA

CUE 0.91** 0.87** 0.98** 0.97** 0.90** 0.75 ns

0.56 ns
0.95** 0.90* )0.99**
DW 0.98** 0.97** 0.94** 0.77ns 0.96** 0.84** 0.99** 0.99** )0.91**
LA 0.92** 0.86* 0.64ns 0.93** 0.89** 0.97** 0.95** )0.85*
SLA 0.99** 0.89* 0.87* 0.69ns 0.99** 0.97** )0.98**
Chla-a 0.94** 0.83* 0.61ns 0.96** 0.95** )0.99**
RWC 0.63ns 0.32ns 0.82* 0.81* )0.93**
MSI 0.93** 0.92** 0.96** )0.75ns
AsA 0.78ns 0.82* )0.54ns
Phen 0.99** 0.95**
Prol 0.90*

CUE, coefficient of uniformity of emergence; DW, seedling dry weight; LA, leaf area; SLA, specific leaf area; Chl-a, chlorophyl-a; RWC, relative
leaf water contents; MSI, membrane stability index; AsA, ascorbic acid; Phen, total soluble phenolics; Pro, leaf free prolines; MDA, malondialde-
hyde; ns, non-significant.
*Significant at P < 0.05; **Significant at P < 0.01.

delayed and erratic stand establishment (Table 2),
decreased the seedling dry weight (Table 2), shoot length,
leaf area, specific leaf area (Table 3), leaf emergence
(Fig. 1) and elongation (Fig. 2) in tested wheat cultivars.
Limited water availability during the imbibition phase of
germination is the primary reason for delayed and erratic
stand establishment (Murillo-Amador et al. 2002). How-
ever, the results of this study indicated improvement in
stand establishment (Table 2), seedling dry weight
(Table 2), shoot length, leaf area and specific leaf area by
seed priming with AsA (Table 3). Seed priming with AsA
substantially improved the stand establishment under
drought and well-watered conditions owing to early com-
pletion of pre-germination metabolic activities during
priming. Modulation of hydrolases during lag phase of
germination by AsA helped to build germination metabo-
lites (Farooq et al. 2006a), resulting in earlier and uni-
form stand establishment (Table 2). This better
germination start also helped in improving the leaf emer-
gence (Fig. 1) and elongation (Fig. 2) under both well-
watered and drought conditions. AsA also enhances the
shoot organogenesis, resulting in better leaf expansion
and plant growth (Stasolla and Yeung 2001).
Although drought decreased the plant water relations
(Table 4), chlorophyll contents and membrane stability
(Table 5), in both wheat cultivars, root length (Table 3),
leaf MDA contents, soluble phenolics, leaf free proline
contents and AsA contents were improved under drought
(Table 6). Nonetheless, seed priming with AsA improved
the plant water relations (Table 4), chlorophyll contents,
membrane stability (Table 5), soluble phenolics, leaf free
proline contents and AsA contents (Table 6) with simulta-
neous decrease in MDA under drought and well-watered
conditions. However, for mitigating the drought effects,
seed priming with AsA was seen as an excellent strategy in
maintaining plant water relation attributes (Table 4) by
the accumulation of osmolytes such as proline (Table 6).
In addition to improvement in chlorophyll-a contents

ª 2012 Blackwell Verlag GmbH, 199 (2013) 12–22 19

Farooq et al.
(Table 5), exogenous application of AsA, through seed
priming, substantially improved its endogenous level
(Table 6), which triggered the accumulation of proline
and phenolics under drought in particular, as is evident
from strong positive correlation of AsA with proline and
phenolics under drought (Table 8).
Proline is one of the most common osmolytes, which
helps in promoting water retention and alleviating the
negative effect of drought on plants (Serraj and Sinclair
2002). There was strong positive correlation of proline
with RWC, seedling dry weight, leaf area, SLA, chloro-
phyll-a and membrane stability under drought (Table 8),
indicating the proline accumulation improved the plant
water status, thus avoiding the oxidative damages
(Table 6) and allowing the leaf expansion (Fig. 2). At cel-
lular level, maintenance of higher water potential means
increasing stomatal conductance under lower water status
(Sellin 2001), which increases root performance for water
uptake (Chimenti et al. 2006) as has been indicated by
the increase in root length by seed priming with AsA
(Table 3). Increased content of intracellular proline thus
increases the plant’s ability to survive under drought
(Taylor 1996). Improved root system plays key role in
plant surviving during drought (Hoogenboom et al.
1987). Improvement in root system with low decrease of
shoot has been regarded as good indicator of drought
resistance (Guoxiong et al. 2002). However, in rest of the
cases, there was no substantial difference between two
wheat cultivars under well-watered and drought condi-
tions (Tables 1–6).
Malondialdehyde production is taken as an index of
ROS-induced oxidative damage (Teisseire and Guy
2000, Zhang et al. 2007). However, plants have evolved
several antioxidative defence mechanisms, including the
production of enzymatic and non-enzymatic antioxi-
dants to reduce ROS-induced oxidative damages (Pos-
myk et al. 2009). Ascorbic acid is one of the most
important ubiquitous non-enzymatic antioxidants pres-
ent in plants (Smirnoff 2000, Smirnoff and Wheeler
2000) with higher concentration in leaves than that in
other plant parts (Smirnoff 2005). AsA detoxifies several
ROS produced during the Mehler reaction (Foyer and
Noctor 2000). Increase in endogenous level of AsA by
seed priming helped in dousing off the ROS levels as
has been indicated by decrease in MDA contents
(Table 6), strong negative correlation between AsA and
MDA and positive correlation between AsA and mem-
brane stability index under drought (Table 8). Phenolics
also possess antioxidant potential in plant cells (Sgherri
et al. 2004, Wahid and Ghazanfar 2006, Wahid 2007).
Seed priming with AsA improved the phenolics
(Table 6), which helped in decreasing the oxidative
damage, as is evident from its positive and negative
20
correlations with membrane stability and MDA con-
tents, respectively, under well-watered (Table 7) and
drought (Table 8) conditions.
In conclusion, drought has adverse effects on the seed-
ling emergence, stand establishment, allometry and water
relation of plants. However, seed priming with AsA
improves the drought resistance in wheat through
increase in endogenous AsA contents, antioxidant poten-
tial and osmotic adjustment. Manipulation of endogenous
AsA levels through genetic or biotechnological means
may result in the development of drought resistance in
wheat.
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