AJCS 6(7):1199-1203 (2012) ISSN:1835-2707

Induction of salt tolerance in tomato (Lycopersicon esculentum Mill.) seeds through sand
priming

Aamir Nawaz1,2, Muhammad Amjad3, Muhammad Muzammil Jahangir3, Samiya Mahmood

Khan2, Huawei Cui 1, Jin Hu 1*
1
Seed Science Center, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310029, China
2
College of Agriculture Bahauddin Zakariya University, Multan Pakistan
3
University of Agriculture Faisalabad Pakistan
*
Corresponding author: jhu@zju.edu.cn

Abstract

Effect of sand priming on vigour and biochemical changes of two tomato varieties, 205 and 206, at various salinity levels (0, 50, 100
and 150 mM NaCl) were studied. Both varieties differ in their salt tolerance capability and are commonly raised in Zhejiang
province. Seeds were mixed with sand particles diameter ranged between 0.5 mm to 2 mm containing 4 % (v/w) water, sealed in
plastic box, and then were primed at 25 ºC for 72 h. Final germination percentage (FGP), germination index (GI), vigour index (VI),
root length, shoot length, fresh weight and dry weight were studied as tomato seed vigour markers in relation to malondialdehyde
(MDA) contents, catalase (CAT), peroxidase (POD), superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities. Sand
priming significantly improved FGP, GI, VI and seedling vigour attributes of both tomato varieties under salinity stress. Moreover
sand priming treatments significantly enhanced the activities of CAT, POD, SOD and APX whereas reduced the accumulation of
MDA contents under salt stress condition. Our results suggested that sand priming can serve as a promising method to enhance
tomato seed vigour under salt stress condition probably through augmentation of antioxidant enzymes activities. Generally, var 206
responded better compared with var 205 at higher salinity stress.

Keywords: tomato, antioxidant enzymes, salinity, seed vigour.

Abbreviations: CAT, Catalase. POD, Peroxidase. SOD, Superoxide dismutase, APX, Ascorbate peroxidase. MDA,
Malondialdehyde.

Introduction

Tomato is a widely distributed annual vegetable crop which and Fernandez-Munoz, 1999). Seed priming (controlled
is consumed fresh, cooked or after processing by canning, hydration followed by redrying) has been used to reduce
making into juice, pulp, paste, or as a variety of sauces; being germination time, harmonize germination, improve
a rich source of phytochemicals such as lycopene, β- germination rate and improve the crop establishment in many
carotene, flavonoids, vitamin C and essential nutrients crops under stress conditions. These priming treatments
(Beutner et al., 2001). Abiotic stresses are major constraints which enhance seed germination include hydropriming
for global crop production. Among various abiotic stresses, (Afzal et al., 2002) osmopriming (Hardegree and Van Vactor,
salinity has become a severe threat to ensure food security by 2000; Rouhi et al., 2011), solid matrix priming (Ghassemi-
affecting about one-third of the irrigated land on earth Golezani et al., 2010) hormonal priming halopriming (Afzal
(Mengel et al., 2001). Salt stress limits plant growth and et al., 2009; Nawaz et al., 2011) sand priming (Hu et al,
productivity, mainly by inducing osmotic effects, ion-specific 2006). The beneficial effect of priming has been associated
effects and oxidative stress (Okhovatian-Ardakani et al., with various biochemical, cellular and molecular events
2010). Reactive oxygen species (ROS) attack proteins, lipids including synthesis of DNA and proteins (Bray et al., 1989).
and nucleic acids, and the degree of damage depends on the Priming is also thought to increase activity of many enzymes
balance between formation of ROS and its removal by the and thus counteracts the effects of seed ageing (Lee and Kim,
antioxidative scavenging systems and it appears to represent 2000). Priming treatment significantly enhanced the activities
an important stress-tolerance trait. Elimination of ROS is of catalase (CAT), peroxidase (POD), superoxide dismutase
mainly achieved by antioxidant compounds such as ascorbic (SOD) and soluble sugar content and reduced the
acid, glutathione, thioredoxine and caroteniods, and by ROS malondialdehyde (MDA) accumulation under the salt stress
scavenging enzymes e.g., superoxide dismutase, glutathione condition in the seedlings (Hu et al., 2006). The faster growth
peroxidase and catalase (Noctor and Foyer, 1998). Salinity of tomato plants from primed seeds seems to be the result of
reduces tomato seed germination and lengthens the time higher capacity for osmotic adjustment because plants from
required for germination to such an extent that the primed seeds have more Na+ and Cl- in roots and more sugars
establishment of a competitive crop by direct seeding would and organic acids in leaves than plants from non-primed
be difficult in soils where the electrical conductivity of a seeds (Cayuela et al., 1996). Priming with polyethylene
saturated extract was equal to or above 8 dS m -1 (Cuartero glycol (PEG) has been applied in vegetable seeds at present;
however, it is not economical for the poor farmers because of

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its high cost. Sand as a priming matrix can be used as an higher final germination, germination index, vigour index
alternative which is simple, highly effective and affordable and increased biomass of primed seeds as compared to
for resource poor farmers an ideal priming medium needs to unprimed seeds of both tomato varieties under varying
attain some characteristics i.e inert media, easy separation salinity environments (Table 1). This earlier synchronized
from seeds, no damage to seeds and high ability of holding and faster germination can be attributed to the enhanced
water. Sand as a priming medium satisfies above mentioned synthesis of DNA, RNA and proteins during priming
conditions. Very little works on sand priming in field crops operations (Bray et al., 1989). Significant enhancement in
has been reported yet (Hu et al., 2002; Hu et al., 2006). final germination after sand priming could possibly be the
However, scanting information is available for usage of sand aftermath of an array of physiological processes e.g. reserves
priming to improve tomato seed germination under salt stress food material breakdown, increased cell division and
conditions in relation to antioxidant defence mechanism. expansion of embryonic axis etc. Moreover this earlier and
Therefore, the objective of present investigation was to synchronized germination of sand primed seeds could be
explore benefits (if any) of sand priming to enhance tomato ascribed to increased metabolic activities in sand primed
seed germination through modulation in antioxidant enzyme seeds as compared to unprimed seeds. Salinity stress inhibits
activities under salt stress. overall plant growth. However, normally root length is more
influenced than shoot length (Jamil et al., 2006). Shoot and
Results root lengths increased with the application of different sand
priming treatments under salinity stress (table 1). This
Germination and seedling vigour evaluation increased shoot and root lengths as compared to high salt
stress may be due to enhanced cell wall extensibility of the
Salinity stress significantly reduced final germination;
primed seeds. Higher fresh and dry weights are reported to
however, sand priming improved final germination of both
correlate with the earlier start of germination. Resultant
tomato varieties. Final germination of both varieties was
increased fresh and dry weights in sand primed seeds are in
significantly increased when treated with sand priming at
conformity with the findings of earlier researchers
various levels of salinity stress (Table 1). Sand priming
(Chookhampaeng et al., 2008). The improvement in seedling
improved germination and vigour index in both varieties at
fresh and dry weights might possibly be the out come of
all salinity levels. While root (2.53 cm) and shoot lengths
increased cell division within the apical meristem induced by
(1.26 cm) of var 205 were significantly improved after sand
sand priming operations.Salinity can trigger oxidative stress
priming in combination with high salt stress (150 mM) as
in plant tissues through increased production of reactive
compares to high stress salt (150 mM) treatments alone.
oxygen species, which can induce membrane lipid
Whereas seeds primed with sand exhibited more shoot fresh
peroxidation. So, accumulation of malondialdehyde can
weight (12.83 mg and 26.33 mg) and dry weight (2.50 mg
serve as an important oxidative stress indicator. MDA
and 5.03 mg) in 205 and var 206 respectively under high salt
contents are reported to increase with the increase in salt
stress (150 mM) (Table 1).
stress in various horticultural crops (Khan and Panda, 2008).
The reduction of MDA accumulation in the seedlings of sand
Antioxidant enzyme activities and malondialdehyde primed seeds might be associated with better membrane
contents repair during sand priming process and inductive responses
of antioxidant enzymes which can provide protection against
The results shoed that the MDA content in shoots of both
oxidative damage (Fig. 1e). In order to combat reactive
tomato varieties was significantly reduced after sand priming
oxygen species which augment salinity induced oxidative
in comparison to high-salt stress of (150 mM). The activity of
stress plants have developed some particular mechanisms
antioxidant enzymes was significantly changed after
such as induction of antioxidant enzymes (SOD, CAT, POD,
exposure to high salt stress, CAT, POD, SOD and APX
APX etc) and non-enzymatic antioxidants (ascorbic acid and
activities increased in sand primed seeds of both tomato
reduced glutathione etc). Similarly in our study significant
varieties (Fig 1). At the same time, the differences between
increases in activities of some antioxidant enzymes such as
control and sand primed seedlings were considerable. The
SOD, CAT, POD, APX in sand primed seeds in combination
activities of CAT increased (4.5 %) POD (15.3 %), SOD (6.6
with salt stress as compared to only salt stressed seeds in both
%) and APX (4.1 %) after sand priming in combination with
tomato varieties was noticed. Such induction of antioxidant
high salt stress in var 206 as compared with var 205 (Fig 1).
enzymes might have triggered inherent antioxidant defense
Maximum CAT activity was recorded in sand priming in
mechanism in the sand primed seeds which might be
combination with 50 mM NaCl followed by sand priming in
responsible for rejuvenation of vigour in tomato (Fig. 1).
combination with 100 mM NaCl, whereas an increased
Such enhancement of seed germination through priming has
activity of POD was recorded in sand priming at 50 mM
also been associated with stimulation of antioxidant activities
NaCl followed by 100 mM NaCl in var 206. Meanwhile the
in various crops by previous researchers (Bailly et al., 1997;
highest SOD and APX activity was recorded in seedlings
Chiu and Sung, 2002).
raised from sand priming at 50 mM NaCl and 100 mM NaCl
in var 206 compared with var 205.
Materials and methods
Discussion
Sand priming
Priming hastens and synchronizes seedling emergence and is
Seeds of two tomato varieties ‘205’ and ‘206’ were obtained
capable of enhancing seedling tolerance to biotic and abiotic
from Zhejiang Academy of Agricultural Sciences.198,
stresses including salinity stress during the critical phase of
Shiqiao Rd, Hangzhou, Zhejiang, P, R, China. The reason for
seedling establishment and consequently can ensure uniform
choosing these two tomato varieties is that they vary in salt
crop stand and yield improvement (Afzal et al., 2009). Sand
tolerance capability and are under common cultivation in the
priming significantly improved germination potential and
Zhejiang province with initial moisture contents of 8.23 %
stand establishment of both tomato varieties as indicated by
and 8.13 %, respectively. Sand particle diameter ranged

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 Table 1. Effect of sand priming on germination and seedling vigour of two tomato varieties 205 and 206 in response to different concentrations
of NaCl.
Variety Priming treatment FGP (%) GI VI Root length Shoot length Fresh wt Dry wt
(cm) (cm) (mg/plant) (mg/plant)
205 Control 84.66 ab* 52.28 bc 174.12 bc 4.93 c 3.32 ab 39.13 b 10.53 b
Sand priming 94.33 a 93.67 a 324.68 a 5.60 a 3.62 a 41.53 a 11.96 a
50 mM NaCl 76.66 b 46.83 c 160.57 c 5.43 ab 3.39 ab 32.30 c 6.80 d
100 mM NaCl 15.10 d 4.86 e 15.59 e 4.96 c 3.26 ab 28.56 d 6.70 d
150 mM NaCl 2.20 d 0.22 e 0.04 e 0.10 e 0.06 d 4.5 f 1.20 f
SP+ 50 mM NaCl 85.00 ab 57.25 b 195.76 b 5.33 ab 3.41 ab 38.96 b 9.30 c
SP+ 100 mM NaCl 50.20 c 21.52 d 67.57 d 5.10 bc 3.13 b 31.00 c 6.83 d
SP+ 150 mM NaCl 9.33 d 0.78 e 0.97 e 2.53 d 1.26 c 12.83 e 2.50 e
LSD at 0.05 6.583 3.251 16.145 0.159 0.207 0.842 0.556
206 Control 87.10 a 58.49 c 174.23 b 5.16 a 2.98 b 44.67 a 15.06 a
Sand priming 94.66 a 102.11 a 312.32 a 5.36 a 3.06 ab 41.63 b 11.90 b
50 mM NaCl 68.46 b 36.36 d 102.19 c 5.03 ab 3.04 ab 35.40 c 8.16 d
100 mM NaCl F 5.33 de 1.086 e 3.4033 d 4.63 b 3.13 ab 31.63 d 7.23 e
150 mM NaCl i 0.00 e 0.00 e 0.00 d 0.00 d 0.00 d 0.00 f 0.00 g
SP+ 50 mM NaCl g 87.11 a 81.20 b 270.24 a 5.20 a 3.36 a 42.36 b 10.20 c
SP+ 100 mM NaClu 40.46 c 38.14 d 120.26 c 5.10 a 3.16 ab 33.43 d 6.80 e
SP+ 150 mM NaClr 17.76 d 13.15 e 21.68 d 3.06 c 1.60 c 26.33 e 5.03 f
LSD at 0.05 7.062 7.182 19.88 0.207 0.173 0.859 0.366
* Small letters behind data mean differences between treatments of the same variety in the same column (α=0.05, LSD). FGP = Final germination
percentage, GI = Germination index, VI = Vigour index. SP = Sand priming.

between 0.5 mm to 2 mm with coherence nil to very slight, Measurement of antioxidant enzyme activities and
cannot be moulded, single grains adhere to fingers, nil to malondialdehyde contents
slight turbidity when puddled. The sand was washed with
water and then dried at 130 °C for 4 h. Each gram of tomato Whole seedlings with fourteen-day-old of tomato were used
seed was mixed with 40 g of sand containing 4 % (v/w) water according to the method of guaiacol to measure the
and sealed in plastic box. Tomato seeds were primed at 25 °C antioxidant enzyme activities and contents of
for 72 h and after priming were separated from sand and malondialdehyde (Zhu and Zhong, 1990). About 0.3 g
dried at room temperature for 24 h. In this experiment, the sample was homogenized in 4 ml extraction buffer consisting
seeds which were used as control (CK) were not sand primed. of 50 mM phosphate (pH 7.8). This homogenate was
They were used as such without any priming treatment. centrifuged at 4 °C for 20 min at 12,000 rpm and the
resulting supernatant was used for determination of various
Germination and seedling vigour evaluation enzymes activity and MDA contents by using a
spectrophotometer (UV-2450, SHIMADZU, Japan).
Saline solutions of 0, 50, 100 and 150 mM NaCl
CAT activity was measured by reduction in absorbance at
concentrations were prepared. The primed and unprimed
240 nm due to the decline of extinction H2O2. The 3 ml
seeds (CK) were placed in 9 cm diameter petri dishes
reaction mixture containing 2.8 ml phosphate buffer (25 mM,
containing two layers of moistened blotters with 4 ml of
pH 7.0), 0.1 ml H2O2 (0.4 %) and 0.1 ml enzyme extract was
respective saline solutions. After that, seeds were kept in a
used. The reaction started with the addition of H2O2 (Cakmak
germination chamber at 25 °C under alternating cycle of 12 h
and Marschner, 1992). The enzyme activity was calculated in
of light and 12 h darkness for 10 days. In the first 4 days, 1
terms of l mol of H2O2 g-1 FW min-1 at 25 ± 2 °C.
ml 0.8 % NaCl solution was supplied in the petri dishes.
Three replicates of 75 seeds each for each treatment were
POD activity was measured with guaiacol as the substrate in
used. Seeds were considered germinated when a 2 mm long
a total volume of 3 ml (Zhang, 1992). The 3 ml reaction
radicle protruded through the seed coat. Final germination
mixture consisted of 2.7 ml phosphate buffer (25 mM, pH
percentage was calculated on the tenth day after sowing.
7.0), 0.1 ml guaiacol (1.5 %), 0.1 ml H2O2 (0.4 %) and 0.1
ml of enzyme extract. Increase in the absorbance due to
Germination index (GI=  Gt
Tt ,
oxidation of guaiacol (E = 25.5 mM-1 cm-1) was measured at
470 nm. The enzyme activity was calculated in terms of l mol
of guaiacol oxidized g-1 FW min-1 at 25 ± 2 °C.
where Gt is number of germinated seeds in the time t day, Tt
is time corresponding to Gt in days) was calculated as SOD activity was assayed by measuring its ability to inhibit
described by (Hu et al., 2006). the photochemical reduction of nitroblue tetrazo-lium (NBT)
Tomato seedlings were harvested after two weeks and (Rao and Sresty, 2000). NBT reaction solution contained 50
washed with deionized water. These seedlings were initially mmol L-1 phosphate buffer (pH 7.8), 13 mmol L-1
used to measure root and shoot lengths and later on used for methionine, 75 μmol L-1 NBT, 2 μmol L-1 riboflavin, 0.1
fresh and dry weight measurement. Dry weight was mmol L-1 EDTA. The reaction mixture was 3.1 ml, which
determined after oven drying the samples at 80 °C for 24 h. contained 3 ml NBT reaction solution and 0.1 ml of enzyme
extract. Reaction was started by adding 2 μmol L-1 riboflavin

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Fig 1. Effect of sand priming on (a) Catalase (CAT) (b) Peroxidase (POD) (c) Superoxide dismutase (SOD) (d) Ascorbate peroxidase
(APX) activities and (e) Malondialdehyde (MDA) contents in leaves of two tomato varieties 205 and 206. Data represents the
average of three replicates. SP means sand priming and CK means control.

and placing the reaction tubes under 15 W fluorescent lamps Data analysis
for 15 min. A complete reaction mixture without enzyme
extract served as a control. The photoreduction of NBT was The main effect of salt treatment (from both NP- and Sand
measured at 560 nm and one unit of SOD was defined as primed-seeds) was tested by one-way analysis of variance
being present in the volume of extract that caused inhibition (ANOVA). Percentage data were arcsin-transformed before
of the photo- reduction of NBT by 50 %. analysis according to ŷ = arcsin [sqr (x/100)]. Means were
compared between treatments by LSD (least significant
APX activity was measured according to Nakano and Asada difference) at the 0.05 confidence level using Statistix 8.1
(1981). The assay depended on the decrease in absorbance at software (Copy right 2005, Analytical Software, USA).
290 nm as ascorbate was oxidized. The 3 ml reaction mixture
consisted of 2.7 ml phosphate buffer (25 mM, pH 7.0), 0.1 ml Conclusions
ascorbate (7.5 mM), 0.1 ml H2O2 (0.4 %) and 0.1 ml of
enzyme extract. The reaction started by addition of H2O2. Our data suggests that sand priming can serve as a promising
The enzyme activity was calculated in terms of l mol of AsA tool for alleviation of salt stress in tomato through enhancing
g-1 FW min-1 at 25 ± 2 °C. seedling vigour and triggering of antioxidant enzymes
defense response.
MDA concentration was determined as 2-thiobarbituric acid
(TBA) reactive metabolites (Zhang et al., 2007). About 1.5 Acknowledgements
ml extract was homogenized in 2.5 ml of 5 % TBA made in 5
% trichloroacetic acid (TCA). The mixture was heated at 95 The research was supported by Higher Education
°C for 15 min, and then quickly cooled on ice. After Commission of Pakistan, key project of Natural Science
centrifugation at 5,000 g for 10 min, the absorbance of the Foundation of Zhejiang Province (No. Z3100150) and
supernatant was measured at 532 nm. Correction of non- Special Fund for Agro-scientific Research in the Public
specific turbidity was made by subtracting the absorbance Interest (201203052), P. R. China.
value measured at 600 nm. The concentration of MDA was
calculated in terms of n mol of g-1 FW.

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