Acta Physiologiae Plantarum (2020) 42:139

https://doi.org/10.1007/s11738-020-03124-x

ORIGINAL ARTICLE

Seed priming with gibberellic acid rescues chickpea (Cicer arietinum L.)

from chilling stress
Tariq Aziz1   · Erkut Pekşen1

Received: 5 March 2019 / Revised: 6 July 2020 / Accepted: 13 July 2020

© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2020

Abstract
Chickpea is sensitive to low temperature stress, especially during germination and stand establishment. Present study was
planned to rescue the chickpea seedlings from chilling stress through seed priming with gibberellic acid. A study compris-
ing of two chickpea cultivars (cv.) viz. Çağatay (a chilling tolerant cultivar: CTC) and Akçin (a chilling sensitive cultivar:
CSC) in combination with 0, hydropriming, 5 µM, 10 µM, 15 µM, 20 µM gibberellic acid ­(GA3) seed priming treatments was
tested in completely randomized design under chilling temperature. Primed and unprimed seeds were sown at 9 ± 0.5 °C day
temperature for 14 h and 7 ± 0.5 °C night temperatures for 10 h. Final emergence percentage (FEP) in both cultivars was
noted higher in 10 µM G ­ A3 seed priming. Coefficient of uniformity of emergence (CUE) was increased and time taken to
50% emergence (­ E50) was shorten with the application of 5 or 15 µM G ­ A3. Mean emergence time (MET) was reduced in
both cultivars in 20 µM ­GA3 seed treatment. Emergence energy (EE) and emergence index (EI) of CTC were increased in
15 µM ­GA3. In CSC 5 µM G ­ A3, seed treatment was most productive treatment under low temperature. Higher doses of G ­ A3
seed treatments in CSC were proved very effective in maintaining high relative water contents and low electrolyte leakage.
Plant height, root length and number of flowers were also increased in ­GA3 primed treatments. In conclusion, seed priming
with ­GA3 can be used in chickpea for good stand establishment, crop growth, reducing electrolyte leakage and maintaining
high relative water contents.

Keywords  Gibberellic acid · Chilling stress · Chickpea · Seed priming · Cultivars

Introduction inhibit hydrolytic enzymes activities in germinating seeds.

Hydrolytic enzymes convert complex food reserve to the
Chickpea ranks third among food grain legume crops in simple useable form for embryo growth during seed germi-
the world. Although, it is a member of cool season legume nation (Szopińska and Politycka 2016). Chilling can disrupt
crops, but gives poor response to low temperatures (Singh the function of cellular membrane and disturb the physio-
et al. 1993). Normally, chickpea requires 21–29 °C tempera- logical and biochemical processes. Cell membranes are sites
tures during day and 20 °C during night time for optimum considered to be the primary targets of hormonal actions
growth and development (Kulkarni and Chimmad 2014). in plants to perform different processes (Trewavas and Gil-
Low temperature adversely affects its growth and develop- roy 1991). These sites are very vulnerable to environmental
ment (Croser et al. 2003). The seed germination phase is rel- stresses (Kuiper 1985; Lyons 2012). Stability of membrane
atively more sensitive to chilling stress. The performance of constituents, particularly lipids are very important in main-
lateral growth stages is associated with competence of seed taining membrane integrity and cell functionality (Mazliak
germination (Farooq et al. 2017). Chilling can slowdown or 1983; Leshem et al. 1990). Huge changes in membrane’s
lipid composition occurred in higher plants to maintain
chlorophyll activity during chilling stress (Pham et al. 1982,
Communicated by J. Gao.
Quartacci et al. 1995). Such changes might be the result of
* Tariq Aziz an increase of unsaturated fatty acids ratio in the galactolip-
tariqbwp@gmail.com ids. It decreases the temperature of transition phase of the
total thylakoid lipid (Moon et al. 1995), resulting in higher
1
Department of Field Crops, Faculty of Agriculture, Ondokuz membrane stability when temperature touches lower critical
Mayıs University, 55139 Samsun, Turkey

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levels. It has been reported that the impact of chilling is Pots were filled with that well homogenized mixture. Three
not restricted to germination it may also change nutrients pots were excessively irrigated and left overnight allow to
dynamics, water uptake, interrupt exchange of gases and draining out extra water to determine the field capacity by
photosynthesis (Yadav 2010), thereby, reducing plant growth calculating the actual amount of water hold by the soil mix-
and yield. Low temperature directly effects the photosyn- ture. Primed and unprimed seeds were sown in pots. After
thetic machinery by disturbing thylakoid membranes that is sowing, pots were irrigated with calculated amount of water
the main site of light reactions during photosynthesis (Zhou to bring them at field capacity. After irrigation, pots were
et al. 2007). In chickpea, poor initial crop stand increases shifted to growth room by setting growth room temperatures
the chances of chilling vulnerability; therefore, can cause at 9 ± 0.5 °C day: 7 ± 0.5 °C night. Photoperiod was set to
considerable damage and yield losses (Croser et al. 2003). 14 h and 10 h for day and night, respectively. Light intensity
Gibberellic acid is a growth hormone involved in seed was set at 350 mE ­m2 ­s1 photon flux density.
germination. Earlier research findings demonstrated that
low-temperature stress harmed seedling emergence in rice Seed priming
but seed treatment with ­GA3 significantly increased seed-
ling emergence (Chen et al. 2005). In many other studies, it Seed priming was carried out by dipping the seeds in
has been reported that ­GA3 improves abiotic stress tolerance well aerated solution of gibberellic acid consisting of 0
in chickpea (Kaur et al. 1998). Seed priming with growth (hydropriming or HP), 5, 10, 15 and 20 µM for 4.5 h at
hormones or salts is one of the pragmatic methods used to 23 ± 2 °C. Seed weight to solution volume ratio was kept
improve seed germination and seedling establishment under 1:5 (w/v) (Farooq et al. 2006). After each seed priming treat-
chilling stress (Farooq et al. 2017). Up to now, many studies ment, seeds were surface washed with running tap water and
have been reported about the progressive role of seed prim- placed in the shade under forced air at 23 ± 2 °C, until initial
ing in developing tolerance in plants (Farooq et al. 2008) moisture level was achieved. Finally, seeds were packed in
but the role of G­ A3 via seed priming under chilling stress plastic bags and kept in a cool place at 4 °C till sowing (Lee
still not investigated. So, using seed priming to overcome and Kim 2000). Seeds without any treatment were consid-
chilling stress could be one of possible options to grow up ered as a control treatment.
crops under abnormal temperatures. In the current research,
we studied the role of seed priming with gibberellic acid to
improve chilling tolerance in chickpea during seed germina- Stand establishment and seedling vigour
tion and early growth stages under controlled conditions. evaluation

The number of germinated seeds was calculated every day

Materials and methods according to the protocol described in seedling evaluation
Handbook of Association of Official Seed Analysis (1990).
Plant material Time taken to 50% emergence ­(E50) was measured following
the equation described by Coolbear et al. (1984), and revised
Seeds of chickpea cultivars were obtained from Black Sea by Farooq et al. (2005). Mean emergence time (MET) was
Agricultural Research Institute, Samsun, Turkey, and were determined using the formulae of Ellis and Roberts (1981).
used as an experimental material. The selection of the cul- The energy of emergence (EE) was calculated according to
tivars was made on the bases of our own earlier screening Farooq et al. (2008). The coefficient of uniformity of emer-
experiments. Out of 24 Turkish released chickpea cultivars, gence  (CUE) was measured by applying the equation of
two cultivars (Çağatay and Akçin) were selected for further Bewley and Black (1994). The emergence index (EI) was
study. Çağatay cultivar was known to be chilling tolerant measured as described in the association of official seed
cultivar (CTC) and Akçin cultivar to be chilling sensitive analysis (AOSA 1983). Seedling dry weight was determined
cultivar (CSC). after drying the plant samples at 70 °C for 72 h days.

Method Measurement of electrolyte leakage

The experiment was conducted in plastic pots. The dimen- Membrane stability was estimated on the bases of electrolyte
sions of the pots were comprising of 22 cm width × 28 cm in leakage following the protocol of Blum and Ebercon (1981).
height having capacity of carrying 9 kg soil. The soil used in Leaf segments, weigh 0.5 g were rinsed with distilled water
the pots was passed through 5 mm sieve to get off all coarse and immersed in test tubes having 10 ml distilled water. Heads
materials out. Sieved soil was mixed with peat soil and of all test tubes were covered with aluminum foil and placed
farm yard manure by keeping 1:1:1 ratio of each material. in a water bath (NÜVE BS 302) at 40 °C for 30 min. All the

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Acta Physiologiae Plantarum (2020) 42:139 Page 3 of 10  139

test tubes were taken out and kept at room temperature to bring chickpea. The state of crop establishment and the level of
their temperature down to 25 °C, followed by measuring ­EC1 its productivity is related to the vigorous seed germination.
of the test tubes with a conductivity meter (Delta OHM HD Chilling stress has many adverse effects on seed germina-
8706). After measuring ­EC1 of the all test tubes containing tion and stand establishment. In our study, chilling stress
same leaf samples were again placed in water bath for 10 min reduced final emergence percentage (FEP) but seed priming
at 100 °C. Samples were taken out and cool down to 25 °C. with 10 μM ­GA3 improved FEP in both cultivars followed
The electrical conductivity of killed tissues ­(EC2) was meas- by 5 μM G ­ A3 seed treatment in CTC (Fig. 1a). CSC showed
ured. Electrolyte leakage was calculated as the ratio between lower FEP than that of CTC. Emergence energy (EE) was
­EC1 and ­EC2. recorded higher in CTC in combination with 15 μM ­GA3
seed priming while EE of CSC was recorded higher in 5 μM
Relative water content (RWC) ­GA3 seed treatment (Fig.  1b). Although, chilling stress
delayed time taken to 50% emergence ­(E50) and mean emer-
Fresh leaves were used to measure the relative water contents. gence time (MET) in both cultivars but seed priming with
Leaf samples (0.5 g; ­Wf) were floated on distilled water in ­GA3 reduced E ­ 50 and MET. E­ 50 and MET were distinctly
Petri dishes for the period of 2 h to absorb water. After due higher in untreated control treatment as compared to the
time, saturated leaves were taken out and were surface dried ­GA3-treated seeds. Minimum ­E50 and MET were recorded in
with soft tissue paper. The saturated weight ­(Ws) were taken seed priming with 10 and 15 μM ­GA3, respectively (Fig. 2a,
and then dried the leaves samples for 24 h at 80 °C in oven for b). The data related to the comparison of coefficient of uni-
determining the dry weight ­(Wd) (Jukanti et al. 2012). formity of emergence (CUE) showed that both cultivars
Relative water content (RWC) was calculated by the fol- differ significantly with each other. G
­ A3 seed treatments at
lowing equation (Barr and Weatherley 1962): the rate of 5 and 15 μM increased CUE in CSC and CTC,
RWC (%) = [(Wf − ­Wd)/(WS − ­Wd)] × 100 respectively. Likewise, emergence index (EI) was recorded
higher in CSC in 15 μM ­GA3 seed treatment. In CTC, HP
Phenological parameters improved EI as compared to other seed priming treatments
(Fig. 3a, b).
Phenological parameters like time taken from sowing to first
flowering (days), time taken from sowing to 50% flowering
(days), time taken from sowing to 100% flowering (days) (a) CTC CSC
100 a ab a
and time taken from sowing to physiological maturity (days) 90 bcd
bc
bcd
ab ab ab bc
d cd
were considered when almost 50% of plants reached to their 80
70
particular stage. 60
FEP (%)

50
Statistical analysis 40
30
20
Experiments were conducted in a completely randomized 10
0
design (CRD) with factorial arrangement using four repli- Control HP GA 5µM GA 10µM GA 15µM GA 20µM
cations. For the comparison of treatment means, standard Seed priming treatments
errors were figured out using Microsoft Excel program. Data CTC CSC
from the study were analysed using ‘Statistix 10′ window 100 (b) abc
ab ab ab
a
ab
abc bc bc
bc
program at 0.05% probability level. Means showing statisti- 80
cd
d
cally significance were separated by LSD test at p < 0.05
60
and mean separation were shown on the columns on the
EE (%)

graphs for each parameter. Graphical presentation of means 40

with error bars based on the standard error of the mean were 20
made using Microsoft Excel program.
0
Control HP GA 5µM GA 10µM GA 15µM GA 20µM
Seed priming treatments
Results
Fig. 1  Influence of pre-sowing gibberellic acid (­ GA3) seed treatments
Stand establishment on a final emergence percentage (FEP) and b energy of emergence
(EE) under chilling stress using four replications. Means with the
same letter are not significantly different (p < 0.05) and bars on the
Low temperature is a major constraint to the seed germi- columns show standard error (± S.E) of the means. Whereas CTC​
nation, stand establishment and early seedling growth of chilling tolerant cultivar, CSC chilling sensitive cultivar

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 139   Page 4 of 10 Acta Physiologiae Plantarum (2020) 42:139

CTC CSC (a) CTC CSC

25 (a) 1.00
abcd
a

a abc ab abc
20 abcde
b 0.75 abcd
bc bcd cde
efgh cde defg cdef bcde
defgh
MET (days)

15 fgh gh h de

CUE
0.50 de e

10
0.25
5

0.00
0 Control HP GA 5µM GA 10µM GA 15µM GA 20µM
Control HP GA 5µM GA 10µM GA 15µM GA 20µM
Seed priming treatments
Seed priming treatments

25 (b) CTC CSC

CTC CSC
10
(b) ab a
20 ab
ab abc
9 a ab abc abc
ab bc bc
8 bc cd bc bcd c bc
cd cd d cd 15 bc
7 cd

EI
E50 (days)

6
10
5
4
5
3
2
1 0
Control HP GA 5µM GA 10µM GA 15µM GA 20µM
0
Control HP GA 5µM GA 10µM GA 15µM GA 20µM Seed priming treatments
Seed priming treatments

Fig. 3  Influence of pre-sowing ­GA3 seed treatments on a coefficient

Fig. 2  Influence of pre-sowing ­GA3 seed treatments on a mean emer- of uniformity of emergence (CUE) and b emergence index (EI) of
gence time (MET) and b time taken to 50% emergence (­ E50) of chick- chickpea cultivars under chilling stress using four replications. Means
pea cultivars under chilling stress using four replications. Means with with the same letter are not significantly different (p < 0.05) and bars
the same letter are not significantly different (p < 0.05) and bars on on the columns indicate standard error (± S.E) of the means. CTC​
the columns indicate standard error (± S.E) of the means. CTC​ chill- chilling tolerant cultivar, CSC chilling sensitive cultivar
ing tolerant cultivar, CSC chilling sensitive cultivar

further increased by pre-sowing seed priming with ­GA3

Periodic shoot length (cm) treatments.

Shoot length (SL) was recorded five times each after Relative water content (RWC) and electrolyte
5 days interval to observe the shoot growth pattern under leakage (EL%)
chilling stress. Influence of ­GA3 on shoot length (SL) is
presented in Fig. 4. Under low temperature stress, SL was Chilling stress resulted decrease in relative water contents
suppressed but seed priming treatment has increased or (RWC) in both cultivars irrespective to seed priming.
maintained SL of both cultivars (Fig. 4). There was dis- The results showed that RWC of chilling tolerant culti-
tinct difference between shoot elongation rate during chill- var were less disturbed under chilling stress as compared
ing period and after shifting the pots to the ambient growth to the chilling sensitive cultivar (Fig. 5a). Seed priming
conditions in the greenhouse. Maximum SL was noted in with ­GA3 effectively improved RWC than that of control
CTC with 10, 15 or 20 μM ­GA3 seed priming as compared treatment. Seed priming with 5 or 10 μM ­GA3 maintained
with control treatment. Although, increasing trend of SL higher RWC in CTC while in CSC seed priming with 15 or
was observed in both but the growth rate of CSC was bit 20 μM ­GA3 treatments were very effective in maintaining
slower than that of CTC under chilling temperature. In higher RWC (Fig. 5a). Chilling stress induced the EL (the
CSC, during initial growth days, the rate of increasing SL solutes come out from the cells), and was recorded higher
was relatively high in 10 and 20 μM ­GA3 pre-sowing treat- in control treatment. The extent of EL is an indicator of
ments but after 07 March, it was preceded by the 15 μM cell membrane damage. Cell membrane is known for one
­GA3 seed treatment. SL of the CSC increased substantially of the swift targets of many plant stressors. Application of
with higher doses (10 or 20 μM ­GA3) of seed treatments ­GA3 proved very effective in reducing electrolyte leakage
as compared with control and other seed priming treat- by stabilizing the cell membrane. Low EL was recorded
ments. CTC grow vigorously and performed better than in 20 μM G ­ A3 followed by 10 μM G ­ A3 seed treatments
that of CSC under chilling stress. Chilling tolerance was

13
Acta Physiologiae Plantarum (2020) 42:139 Page 5 of 10  139

Fig. 4  Role of seed priming 20

with ­GA3 on periodic shoot Control HP GA 5µM GA 10µM GA 15µM GA 20µM
18
length (cm) of two chickpea cul-
tivars sown under chilling stress 16
using four replications. Bars on

Periodic shoot length (cm)

the lines indicate standard error 14
(± S.E) of the means
12

10

0
20-Feb 25-Feb 02-Mar 07-Mar 12-Mar 20-Feb 25-Feb 02-Mar 07-Mar 12-Mar
CTC CS C

80 (a) CTC CSC Chlorophyll contents (μmol ­m−2)

a a a a
70 bc b
Relative water content (%)

de bcd
e
cd cd Chilling stress disordered photosynthesis by lowering and
60 f
disrupting chlorophyll pigments. However, the pretreated
50
seeds with ­GA3 well-maintained chlorophyll contents as
40
compared with the control treatment. Leaf chlorophyll con-
30
tents of CTC and CSC cultivars were recorded higher in
20
15 μM and 5 μM G ­ A3 seed treatments, respectively (Fig. 6a).
10
Chlorophyll contents of the plants after 1 week of their shift-
0
Control HP GA 5µM GA 10µM GA 15µM GA 20µM ing to the greenhouse (ambient environment) revealed higher
Seed priming treatments chlorophyll recovery in CSC as compared with CTC. In
CTC, chlorophyll contents were already higher than CSC
(b) CTC CSC so less change was observed. Seed priming with 20 μM
30
­GA3 was the best treatment for CSC. In CTC, there was no
25 ab
significant difference in chlorophyll contents between seed
Electrolyte leakage (%)

ab a ab ab
ab ab
20 ab priming and control treatment (Fig. 6b).
b
15 b
b

10
Plant growth parameters
5
Pre-sowing ­GA3 seed priming increased shoot dry weight
0
Control HP GA 5µM GA 10µM GA 15µM GA 20µM
significantly in CSC, while there was no significant differ-
Seed priming treatments ence in CTC was found as compared with control treatment.
Seed priming with 5 μM G ­ A3 was the most effective seed
Fig. 5  Role of seed priming with G ­ A3 on a relative water content treatment in increasing plant dry weight (Fig. 7a). Seed
(RWC) and b electrolyte leakage (EL) of two chickpea cultivars sown priming has influenced root dry weight in both chickpea
under chilling stress using four replications. Means with the same let-
ter are not significantly different (p < 0.05) and bars on the columns
cultivars (Fig. 7b). Maximum root dry weight was recorded
indicate standard error (± S.E) of the means. CTC​ chilling tolerant in the CTC sown after 20 μM ­GA3 seed treatment. Similarly,
cultivar, CSC chilling sensitive cultivar seed priming with 10 μM ­GA3 increased root dry weight of
CSC cultivar (Fig. 7b), however, seed priming with 20 μM
­GA3 resulted lowest root dry weight in CSC (Fig. 7b).
indicates that seed priming can be effectively use for ame-
liorating cell membrane damage under low temperature Plant height and root length (cm)
stress. Similarly, our results were promising in reducing
the effect of chilling stress in both cultivars either toler- Seed priming was helpful in improving plant height and root
ance or sensitive (Fig. 5b). length (RL) of both chickpea cultivar. Maximum plant height
was measured in CSC in seed priming with 10 μM G ­ A3

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 139   Page 6 of 10 Acta Physiologiae Plantarum (2020) 42:139

Chlorophyll contents (µmol m-2)

30
(a) CTC CSC
8
(a) CTC CSC

abc a abc a ab
25 ab 7 abcde abcd
ab a b b bcde bcde cde de
b bc e
20 6
b

Shoot dry deight (g)

b bc
15 c c 5

10 4

5 3

0 2
Control HP GA 5µM GA 10µM GA 15µM GA 20µM
1
Seed priming treatments
0
Control HP GA 5µM GA 10µM GA 15µM GA 20µM
Seed priming treatments
60 (b) a
CTC CSC
(b)
Chlorophyll contents (µmol m-2)

50 0.7
b CTC CSC a

bc b 0.6
40
cde cd a
def cd
0.5

Root dry weight (g)

30 f ef
ef ab
f ab ab
0.4 ab ab ab ab ab
20
b
0.3
10 b
0.2
0
Control HP GA 5µM GA 10µM GA 15µM GA 20µM 0.1
Seed priming treatments
0
Control HP GA 5µM GA 10µM GA 15µM GA 20µM
Seed priming treatments
Fig. 6  Role of seed priming with ­ GA3 on a chlorophyll contents
(µmol ­m−2) during chilling stress b chlorophyll contents (µmol m ­ −2)
after chilling stress of two chickpea cultivars sown under chilling Fig. 7  Role of seed priming with ­GA3 on a shoot dry weight b root
stress using four replications. Means with the same letter are not sig- dry weight of two chickpea cultivars sown under chilling stress using
nificantly different (p < 0.05) and bars on the columns indicate stand- four replications. Means with the same letter are not significantly
ard error (± S.E) of the means. CTC​ chilling tolerant cultivar, CSC different (p < 0.05) and bars on the columns indicate standard error
chilling sensitive cultivar (± S.E) of the means. CTC​ chilling tolerant cultivar, CSC chilling
sensitive cultivar

and minimum plant height was in control treatment. Plant

height of CTC varied between 53.66 and 60 cm (Fig. 8a). treatments number of branches were higher in CTC. It means
Root lengths of CSC and CTC were ranged between 6.33 CSC was more responsive to G ­ A3 seed priming in term of
and 16.33 cm and 11.33–17.33 cm, respectively (Fig. 8b). production of branches (Fig. 9a).
Maximum root length was recorded in CTC in 10 μM G ­ A3
seed treatment (Fig. 8b) and the minimum RL was in control
treatment. Overall CTC produced long roots as compared Discussion
with CSC of all priming treatments except seed priming
with 20 μM G ­ A3 where CSC performed better than CTC Chilling temperature stress affected seed germination,
(Fig. 8b). stand establishment and early growth of both tested culti-
vars (Figs. 1, 2, 3). The present findings are in line with
Number of flowers and branches the Yusefi-Tanha et al. (2015). They reported poor seed-
ling emergence under-chilling temperature stress in peas,
Number of flowers were differed in primed and non- but seed priming improved final emergence percentage. The
primed seeds. Seed priming with higher ­GA3 concentration reasons behind low germination were might be the adverse
improved number of flowers in both cultivars (Fig. 9a). Seed effect of reactive oxygen species and poor enzymatic activi-
priming with 10 or 15 μM ­GA3 doses produced higher num- ties. Chilling stress can ramp up the production of reactive
ber of flowers in both cultivars. Maximum number of flow- oxygen species (ROS) which disturbed electron flow during
ers were counted in CTC with 10 μM G ­ A3 seed treatment. metabolism. ROS damage the cell membranes and caused
Number of branches were improved in CSC when seeds electrolyte leakage and proscribe seed germination (Baal-
were sown after seed priming with ­GA3 as compared with baki et al. 1999; Yusefi-Tanha et al. 2019). In seeds, G­ A3
untreated seeds. Overall number of branches were observed involves in physiological and metabolic processes to initiate
higher in CSC in priming treatments while under control seed germination (Pipinis et al. 2012), and also help up to

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Acta Physiologiae Plantarum (2020) 42:139 Page 7 of 10  139

80 (a) CTC CSC

6 (a) a
CTC CSC
a
a a a
70 a ab a
abcd ab ab 5 ab
abc abc abcd ab ab ab
60 bcd bcd
cd b

Number of branches
d b
Plant height (cm)

4
50

40 3

30 2
20
1
10

0 0
Control HP GA 5µM GA 10µM GA 15µM GA 20µM
Control HP GA 5µM GA 10µM GA 15µM GA 20µM
Seed priming treatments
Seed priming treatments

CTC CSC
25 (b) (b)
CTC CSC 20 a
a
abc ab
20 a ab abcd
ab

Number of Fowers
15
ab ab ab bcde cde de
ab bcde
b ab b de
Root length (cm)

b b e
15
10
f

10
5

5 0
Control HP GA 5µM GA 10µM GA 15µM GA 20µM
Seed priming treatments
0
Control HP GA 5µM GA 10µM GA 15µM GA 20µM
Seed priming treatments
Fig. 9  Role of seed priming with ­GA3 on a shoot dry weight and b
root dry weight of two chickpea cultivars sown under chilling stress
Fig. 8  Role of seed priming with ­GA3 on a plant height and b root using four replications. Means with the same letter are not signifi-
length of two chickpea cultivars sown under chilling stress using four cantly different (p < 0.05) and bars on the columns indicate standard
replications. Means with the same letter are not significantly differ- error (± S.E) of the means. CTC​ chilling tolerant cultivar, CSC chill-
ent (p < 0.05) and bars on the columns indicate standard error (± S.E) ing sensitive cultivar
of the means. CTC​ chilling tolerant cultivar, CSC chilling sensitive
cultivar
convert complex food to simple available form in unprimed
alleviate the adverse effect of stress as found in our study seeds for the embryo utilization. Therefore, embryo takes
(Fig. 1a, b). Kirmizi et al. (2010) reported that application much longer time to grow in untreated seeds due to low and
of 150 ppm ­GA3 under low temperature increases germina- slow availability of soluble food.
tion percentage in Pedicularis olympica (Scrophulariaceae). Earlier research findings showed that chilling stress
In the current research, seed priming with ­GA3 improved reduces plant growth but seed treatment with G ­ A3 stimulates
seedling emergence percentage and reduced E ­ 50 and MET plant growth (Rehman and Park 2000). Thakare et al. (2011)
(Fig. 2a, b). Gibberellic acid takes part in inducing hydro- also reported that seed priming with G ­ A3 enhances chick-
lytic enzyme activities such as hydrogenase and α-amylase pea height under low temperature stress. Higher plant height
(Gupta et al. 2013) to initiate germination process in the in ­GA3-treated seedlings, might be the result of higher cell
seed and speed up germination process. Seed priming with division, cell elongation (Naylor 1984), in addition to high
­GA3 can hastens seed germination rate by stimulating and cell wall acid invertase activity (Kaur et al. 1998; Kaya et al.
activating the food-digestive and food mobilizing enzymes 2006). Cell wall acid invertase enzymes catalyze the irre-
(Hartmann et al. 1997). Taiz and Zeiger (2002) reported that versible breakdown of sucrose to free glucose and fructose
embryo naturally releases G ­ A3 that activate particular genes and are the fundamental enzymes take part in controlling
for mRNA transcription by α-amylase during seed germi- cell differentiation and plant development (Koch 1996). ­GA3
nation. The activation of α-amylase and couple of other also trigger the metabolic consumption of soluble sugars to
enzymes degrades the food reserves and makes available form new cell constituents which involves in growth process
for embryo consumption. The other possible reason behind (Jasmine and Merina 2012). In short statured pea (Pisum
good stand establishment was that primed seeds had already sativum) plants, G ­ A3 treatment triggered shoot length by
completed the first two phases of seed germination; in other activating cell-wall invertase enzyme (Wu et al. 1993). In
words, seeds were at the brink of germination. On the other beans (Phaseolus vulgaris), ­GA3-treated plants showed
hand, enzymes must have to digest food reserves first to high invertase activity in elongating internodes (Morris and

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 139   Page 8 of 10 Acta Physiologiae Plantarum (2020) 42:139

Arthur 1985), and resulting stem elongation. G ­ A3 has natu- that externally applied G­ A3 increased quantum yield and
ral character that boost up impaired cell division and cell the ratio of Fv/Fm in chilies. Another study report stated
elongation under unfavorable conditions (Kaya et al. 2006). that ­GA 3 increased chlorophyll content in grape (Lim
This might be the result of less water requirements or high et al. 2004). Seed priming with 200 ppm ­GA3 increased
water up take in the presence of plant growth hormones chlorophyll content in papaya seedlings by accelerating
during seedling growth (Kaur et al. 1998). Chilling stress nitrogen uptake (Ramteke et al. 2016). Under low tempera-
induces production of ROS, causes reduction in cell division ture stress, reduction in chlorophyll contents might be the
and cell elongation (Kaya et al. 2006) and modify biochemi- result of proteolytic enzymes synthesis such as chlorophyl-
cal changes in cell wall during cell growth and restrict cell lase that involved in chlorophyll degradation (Sabater and
wall extension (VanVolkenburgh and Boyer 1985). Rodriquez 1978), and harm the photosynthetic apparatus
Relative water contents give the information about inter- (Yasseen 1983).
nal plant water status and all types of metabolic activities Seed priming with 5 μM ­GA3 was the most effective
are directly linked with the presence of water in the plant seed treatment in increasing plant dry weight (Fig. 7a).
tissues (Flower and Ludlow 1986). RWC is associated with Maximum plant height and root length was measured in
leaf water potential and correlated with plant yield (Lafitte CSC in combination with seed priming with 10 μM G ­ A3
2002). Seed priming with 5 or 10  μM G ­ A3 maintained and minimum plant height was recorded in control treat-
higher RWC in CTC while in CSC seed priming with 15 ment (Fig. 8a, b). Many researchers reported that seed
or 20 μM G ­ A3 treatments were very effective in maintain- priming with ­GA3 accelerates root and shoot length, fresh
ing high RWC (Fig. 5a). Under low temperature stress, low and dry weight and increases leaf water content (Little
RWC were possibly due to the decrease of cell metabolites and MacDonald 2003; Ghodrat and Rousta 2012; Shehzad
and solutes available to keep the water within the cells. The et al. 2014). In another study, no significant change in dry
main effect of low temperature stress on developing seed- matter accumulation was reported in indian grass under
lings were related to turgor loss, as a consequence of turgor chilling stress when the seeds were sown after soaking in
loss, plant showed wilting as well as drought symptoms 1000 ppm ­GA3 solution (Watkinson and Pill 1998). The
(Croser et al. 2003). So, in this regard, high RWC of plant contradictory results in dry matter accumulation in Indian
leaf under low temperature could be used as an indicator of grass might be the seeds required higher concentration of
chilling tolerance (Singh et al. 2012; Ghosh et al. 2016). In ­GA3 to stimulate dry matter accumulation in seedlings. In
20 μM ­GA3-treated seeds low electrolyte leakage occurred sum up, application of seed priming with gibberellic acid
followed by 10 μM ­GA3 seed treatments indicates that seed in chickpea has positive effect on plant shoot and root dry
priming can be effectively used for ameliorating cell mem- weight.
brane damage under low temperature stress (Fig. 5b). Chill- In our study, number of the flowers and branches were
ing stress triggers the production of reactive oxygen species differed in plants grown from primed and non-primed seeds
that disturbed the electron flow across the plasma membrane. with ­GA3 treatment (Fig. 9a, b). Some previous research
Under chilling stress, membrane lipids usually transform investigation showed that seed priming can increased the
from fluid to gel or semisolid or solid states, which limits number of branches from 6.63 to 10.43 per plant (Chavan
membrane permeability (Leshem 2013). Under such condi- et  al. 2014). But in another study, contradictory results
tions, ROS may react with membrane’s polyunsaturated fatty were reported by Khan et al. (2005). ­GA3 involves in flower
acids and induce lipid peroxidation and damage cell mem- induction by increasing invertase enzyme activity in sugar
brane. It might be thought that ­GA3 increases C ­ a2+ ion in the cane (Sacher et al. 1963). In phalaenopsis plants, application
leaf cells which help to keep water balance. Likewise, Ali of ­GA3 up to 1000 ppm increased flower numbers almost
et al. (2012) reported that the role of ­GA3 is obvious in main- double without giving any harm to plants (Cardoso et al.
taining high RWC of leaf. Kaya et al. (2006) also reported 2012). Some other studies revealed that ­GA3 plays a subsidi-
similar results that ­GA3 increase relative water contents by ary role in flower induction (Dong et al. 2017). Our findings
increasing root length and water uptake. In nutshell, seed are agreed with Zhang et al. (2016), they were reported that
priming with ­GA3 proved effective in decreasing EL and ­GA3 regulates flowering in wheat.
increasing RWC under low temperature stress (Fig. 5a, b). On the bases of above study, it is concluded that seed
Chlorophyll contents of CTC and CSC cultivars were priming could help in maintaining stand establishment,
improved with 15 and 5 μM G ­ A3 seed treatments, respec- increasing shoot length, root length, leaf relative water con-
tively (Fig.  6a). Tatar et  al. (2013) noted that chilling tent, seedling dry weight, number of flowers and decreas-
stress inhibit chlorophyll contents, damage its structure ing electrolyte leakage in chickpea cultivars under chilling
and function in non-treated seeds. They also reported that stress. Seed priming could be successfully used for grow-
Sarı-98 and VDI-5 cultivars differ in chlorophyll content ing chickpea in low temperature areas to improve the plant
under chilling conditions. Georgia et al. (2010) reported performance.

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Acta Physiologiae Plantarum (2020) 42:139 Page 9 of 10  139

Author contribution statement  EP and TA discussed the Farooq M, Basra SMA, Ahmad N, Hafeez K (2005) Thermal harden-
research concept and designed the experiment. TA con- ing: a new seed vigor enhancement tool in rice. J Integr Plant
Biol 47:187–193
ducted the experiment, took the data, did the plant and statis- Farooq M, Aziz T, Basra SMA, Wahid A, Khaliq A, Cheema MA
tical analysis which were finally verified by EP. TA prepared (2008) Exploring the role of calcium to improve chilling toler-
the initial manuscript draft which was improved through a ance in hybrid maize. J Agron Crop Sci 194:350–359
series of changes made by EP. Flower D, Ludlow M (1986) Contribution of osmotic adjustment to
the dehydration tolerance of water-stressed pigeon pea (Cajanus
cajan (L.) millsp.) leaves. Plant Cell Environ 9:33–40
Georgia G, Ilias I, Anastasia I, Parthena G (2010) Comparative study
Acknowledgements  The author is thankful to The Scientific and on the effects of various plant growth regulators on growth,
Technological Research Council of Turkey (TÜBİTAK) for awarding quality and physiology of Capsicum annuum L. Pak J Bot
scholarship grant under the BIDEB 2215 program for international stu- 42:805–814
dents. The authors are also thankful to Ondokuz Mayıs University for Ghodrat V, Rousta MJ (2012) Effect of priming with gibberellic acid
providing funding for research project under the program of Scientific ­(GA3) on germination and growth of corn (Zea mays L.) under
and Research Support Funds (PYO.ZRT.1901.15.009). saline conditions. Int J Agri Crop Sci 4:883–885
Ghosh T, Rai M, Tyagi W, Challam C (2016) Seedling stage low
temperature response in tolerant and susceptible rice genotypes
suggests role of relative water content and members of OsSNAC
gene family. Plant Signal Behav 11:e1138192
References Gupta R, Chakrabarty S (2013) Gibberellic acid in plant: still a mys-
tery unresolved. Plant Signal Behav 8:25504
Ali HM, Siddiqui MH, Basalah MO, Al-Whaibi MH, Sakran AM, Al- Harris D, Joshi A, Khan P, Gothkar P, Sodhi P (1999) On-farm seed
Amri A (2012) Effects of gibberellic acid on growth and photo- priming in semi-arid agriculture: development and evaluation in
synthetic pigments of Hibiscus sabdariffa L. under salt stress. Afr maize, rice and chickpea in India using participatory methods.
J Biotechnol 11:800–804 Exp Agric 35:15–29
AOSA (1990) Rules for testing seeds, USA. J Seed Technol 12:1–112 Hartmann HT, Kester DE, Davies FT, Geneve RL (1997) Plant Prop-
Baalbaki RZ, Zurayk RA, Blelk MM, Tahouk SN (1999) Germina- agation, Principles and Practices, 6th edn. Prentice Hall, Upper
tion and seedling development of drought tolerant and susceptible Saddle River, N.J., USA, p 770
wheat under moisture stress. Seed Sci Technol 27:291–302 Jasmine MS, John MA (2012) Effects of gibberellic acid on seed-
Barrs H, Weatherley P (1962) A re-examination of the relative turgid- ling growth, chlorophyll content and carbohydrate metabolism
ity technique for estimating water deficits in leaves. Aust J Biol in okra (Abelmoschus esculentus L. Moench) genotypes under
Sci 15:413–428 saline stress. Res J Chem Sci 2(7):72–74
Bewley J, Black M (1994) Seeds: physiology of development and ger- Jukanti AK, Gaur PM, Gowda C, Chibbar RN (2012) Nutritional
mination. Plenum Press, New York, USA, p 445 quality and health benefits of chickpea (Cicer arietinum L.): a
Blum A, Ebercon A (1981) Cell membrane stability as a measure of review. Br J Nutr T 108:11–26
drought and heat tolerance in wheat. Crop Sci 21:43–47 Kaur S, Gupta AK, Kaur N (1998) Gibberellic acid and kinetin par-
Cardoso JC, Ono EO, Rodrigues JD (2012) Gibberellic Acid (­ GA3) tially reverse the effect of water stress on germination and seed-
on flowering induction and quality of Phalaenopsis orchid. Orna- ling growth in chickpea. Plant Growth Reg 25:29–33
mental Hort 18:135–140 Kaya C, Tuna AL, Alfredo AA (2006) Gibberellic acid improves
Chavan N, Bhujbal G, Manjare M (2014) Effect of seed priming on water deficit tolerance in maize plants. Acta Physiol Plant
field performance and seed yield of soybean [Glycine max (L.) 28:331–337
Merill] varieties. Bioscan 9:111–114 Khan MR, Qureshi AS, Hussain SA, Ibrahim M (2005) Genetic vari-
Chen D, Gunawardena T, Naidu B, Fukai S, Basnayake J (2005) Seed ability induced by gamma irradiation and its modulation with
treatment with gibberellic acid and glycinebetaine improves seed- gibberellic acid in M~ 2 generation of chickpea (Cicer arietinum
ling emergence and seedling vigour of rice under low temperature. L.). Pak J Bot 37:285
Seed Sci Technol 33:471–479 Kirmizi S, Güleryüz G, Arslan H, Sakar HS (2010) Effects of moist
Coolbear P, Francis A, Grierson D (1984) The effect of low tempera- chilling, gibberellic acid, and scarification on seed dormancy in
ture pre-sowing treatment on the germination performance and the rare endemic Pedicularis olympica (Scrophulariaceae). Turk
membrane integrity of artificially aged tomato seeds. J Exp Bot J Bot 34:225–232
35:1609–1617 Koch K (1996) Carbohydrate-modulated gene expression in plants.
Croser J, Clarke H, Siddique K, Khan T (2003) Low-temperature stress: Ann Rev Plant Bio 47:509–540
implications for chickpea (Cicer arietinum L.) improvement. Crit Kuiper P (1985) Environmental changes and lipid metabolism of higher
Rev Plant Sci 22:185–219 plants. Physiol Plant 64:118–122
Dong B, Deng Y, Wang H, Gao R, Stephen GUK, Chen S, Jiang J, Kulkarni M, Chimmad V (2014) Effect of temperature regimes on phe-
Chen F (2017) Gibberellic acid signaling is required to induce nology and yield of chickpea (Cicer arietinum L.). Karnataka J
flowering of chrysanthemums grown under both short and long Agric Sci 27:526–527
days. Int J Mol Sci 18:1259 Lafitte (2002) Relationship between leaf relative water content during
Ellis R, Roberts E (1981) The quantification of ageing and survival in reproductive stage water deficit and grain formation in rice. Field
orthodox seeds. Seed Sci Technol 9:373–409 Crops Res 76:165–174
Farooq M, Hussain M, Nawaz A, Lee DJ, Alghamdi SS, Siddique KH Lee SS, Kim JH (2000) Total sugars, α-amylase activity, and germina-
(2017) Seed priming improves chilling tolerance in chickpea by tion after priming of normal and aged rice seeds. Kor J Crop Sci
modulating germination metabolism, trehalose accumulation and 45:108–111
carbon assimilation. Plant Physiol Biochem 111:274–283 Leshem YL, Cojocaru M, MargelEL-ANI SD, Landau EM (1990) A
Farooq M, Basra SMA, Hafeez K (2006) Seed invigoration by osmo- biophysical study of abscisic acid interaction with membrane
hardening in coarse and fine rice. Seed Sci Technol 34:181–187 phospholipid components. New Phytol 116:487–498

13
 139   Page 10 of 10 Acta Physiologiae Plantarum (2020) 42:139

Lim SC, Kim SK, Kim YH, Youn CK, Yoon T (2004) Vine growth and Singh K, Malhotra R, Saxena M (1993) Relationship between cold
fruit quality of ‘kyoho’ grapes as affected by Mepiquat chloride severity and yield loss in chickpea (Cicer arietinum L.). J Agron
and ­GA3. Acta Hort (ISHS) 653:145–149 Crop Sci 170:121–127
Little C, MacDonald JE (2003) Effects of exogenous gibberellin Szopińska D, Politycka B (2016) The effects of hydro-and osmoprim-
and auxin on shoot elongation and vegetative bud development ing on the germination, vigour and hydrolytic enzymes activity of
in seedlings of Pinus sylvestris and Picea glauca. Tree Physiol common zinnia (Zinnia elegans Jacq.) seeds. Folia Horticulturae
23:73–83 28:3–11
Lyons J (2012) Low temperature stress in crop plants: the role of the Taiz L, Zeiger E (2006) Plant physiology, 4th edn. Sinauer Associates
membrane. Elsevier 4(6):413–444 Inc., Publishers, Massachusetts
Mazliak P (1983) Plant Membrane Lipids: Changes and Alterations Tatar O, Ozalkan C, Atasoy GD (2013) Partitioning of dry matter,
during Aging and Senescence. In: Lieberman M (ed) Post-harvest proline accumulation, chlorophyll content and antioxidant activity
physiology and crop preservation. Nato advanced study institutes of chickpea (Cicer arietinum L.) plants under chilling stress. Bul
series (series a: life sciences), vol 46. Springer, Boston, MA, pp J Agri Sci 19(2):260–265
123–140 Thakare U, Patil N, Malpathak N (2011) Performance of chick pea
Moon BY, Higashi S, Gombos Z, Murata N (1995) Unsaturation of under the influence of gibberellic acid and oxygenated peptone
the membrane lipids of chloroplasts stabilizes the photosynthetic during germination. Adv Biosci Biotech 2(01):40–45
machinery against low-temperature photoinhibition in transgenic Trewavas A, Gilroy S (1991) Signal transduction in plant cells. Trends
tobacco plants. Proce Ntl Acad Sci 92:6219–6223 Genet 7:356–361
Morris DA, Arthur ED (1985) Effects of gibberellic acid on patterns of Van Volkenburgh E, Boyer JS (1985) Inhibitory effects of water deficit
carbohydrate distribution and acid invertase activity in Phaseolus on maize leaf elongation. Plant Physiol 77:190–194
vulgaris. Physiol Plant 65:257–262 Watkinson JI, Pill WG (1998) Gibberellic acid and presowing chilling
Naylor AW (1984) Hormonal regulation of development. II. The func- increase seed germination of Indiangrass [Sorghastrum nutans
tion of hormones from the level of the cell to whole plant. In: (L.) Nash.]. HortScience 33:849–851
Encyclopedia of Plant Physiology, Scott TK (ed) New Se ries. 10: Williams W (1990) Cold-induced lipid phase transitions. Phil Trans R
180–185. Berlin: Springer Verlag Soc Lond B 326:555–570
Pham Thi A, Flood C, da Silva JA (1982) Effects of water stress on Wu LL, Mitchell JP, Cohn NS, Kaufman PB (1993) Gibberellin (GA3)
lipids and fatty acid composition of cotton leaves. Deve Plant enhances cell wall invertase activity and mRNA levels in elongat-
Biol 62:219–224 ing dwarf pea (Pisum sativum) shoots. Int J Plant Sci 154:280–289
Pipinis E, Milios E, Kiamos N, Mavrokordopoulou O, Smiris P (2012) Yadav S (2010) Cold stress tolerance mechanisms in plants. A Rev
Effects of stratification and pre-treatment with gibberellic acid Agron Sustain Dev 30:515–527
on seed germination of two Carpinus species. Seed Sci Technol Yusefi-Tanha E, FallahTadayon SA (2015) Effect of seed priming on
40:21–31 some effective physiological parame- ters on seed germination of
Quartacci MF, Pinzino C, Sgherri CL, Navari-Izzo F (1995) Lipid com- pea (Pisum sativum L.) under chilling stress. J Plant Process and
position and protein dynamics in thylakoids of two wheat cultivars Function 4:1–15
differently sensitive to drought. Plant Physiol 108:191–197 Yusefi TE, Fallah S, Taddayon A (2015) Effect of seed priming on
Ramteke V, Paithankar D, Baghel MM, Kurrey VK (2016) Impact of some effective physiological parameters on seed germination
­GA3 and propagation media on growth rate and leaf chlorophyll of pea (Pisum sativum L.) under chilling stress. J Plant Pro Fun
content of papaya seedlings. Res J Agri Sci 7:169–171 13:1–15
Rehman S, Park IH (2000) Effect of scarification, GA and chilling Yasseen BT (1983) An analysis of the effects of salinity on leaf growth
on the germination of goldenrain-tree (Koelreuteria paniculata in Mexican wheats. Thesis, The University of Leeds, UK
Laxm.) seeds. Sci Hortic 85:319–324 Zhou Y, Lam HM, Zhang J (2007) Inhibition of photosynthesis and
Sabater B, Rodriquez MT (1978) Control of chlorophyll degradation energy dissipation induced by water and high light stresses in rice.
in detached leaves of barley and oat through effect of kinetin on J Exp Bot 58:1207–1217
chlorophyllase levels. Physiol Plant 43:274–276 Zhang N, Xie YD, Guo HJ, Zhao LS, Xiong HC, Gu JY, Li JH, Kong
Sacher J, Hatch M, Glasziou K (1963) Sugar accumulation cycle in FQ, Sui L, Zhao ZW, Zhao SR, Liu LX (2016) Gibberellins regu-
sugar cane. III. Physical and metabolic aspects of cycle in imma- late the stem elongation rate without affecting the mature plant
ture storage tissues. Plant Physiol 38:348 height of a quick development mutant of winter wheat (Triticum
Shahzad R, Khan J, Gurmani AR, Waqas M, Hamayun M, Khan AL, aestivum L.). Plant Physiol Biochem 107:228–236
Kang SM, Lee IJ (2014) Seed priming with gibberellic acid
(GA3) in sponge-gourd modulated high salinity stress. J Life Sci Publisher’s Note Springer Nature remains neutral with regard to
02:75–86 jurisdictional claims in published maps and institutional affiliations.
Singh I, Kumar U, Singh S, Gupta C, Singh M, Kushwaha S (2012)
Physiological and biochemical effect of 24-epibrassinoslide
on cold tolerance in maize seedlings. Physiol Mol Biol Plants
18:229–236

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