Study of dormancy-breaking of Black cumin seeds (Nigella sativa L.)
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Scholars Research Library
Annals of Biological Research, 2012, 3 (6):2651-2655
(http://scholarsresearchlibrary.com/archive.html)
ISSN 0976-1233
CODEN (USA): ABRNBW
Study of dormancy-breaking of Black cumin seeds (Nigella sativa L.)
Hossein Reza Rouhi, Ali Sepehri*, Fatemeh Karimi
Department of Agronomy and Plant Breeding, Faculty of Agriculture, University of Bu-Ali Sina,
Hamedan, Islamic Republic of Iran
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ABSTRACT
This work aimed to examine dormancy breaking of Black cumin seed (Nigella sativa). Seeds collected from the
Isfahan zone in Iran were subjected to different treatments: (a) concentrated sulfuric acid for 30 and 60 seconds, (b)
hot water at 70 and 80°C for 5 and 10 minutes (c) concentrated gibberellic acid for 250, 500, 750, 1000, 1250ppm
for 24h (d) three levels of KNO3 (0.1, 0.2 and 0.3% v/v) and (e) 3 different stratification periods (1, 2 and 3 weeks).
The best percentage of germination, mean germination time, seedling length, seedling dry weight and vigor index
were observed in the case of stratification applied for 3 weeks, but 1 and 2 weeks treatment had no strong effect on
dormancy release same as 3 weeks. In contrast to stratification, seeds showed no response to periods of sulfuric
acid 98% (v/v) and hot water treatments. All of germination traits of Black cumin increased at all concentration of
GA3, but 1250ppm was better than the others. Same as GA3, KNO3 treatment had a positive effect on seed
germination in all levels especially at 0.3% v/v. the effect of GA3 and KNO3 were not higher than stratification in 3
weeks. It was concluded that stratification in 3weeks was more effective treatment on studied traits than 1 and 2
weeks and it was a stronger action on breaking dormancy and germination of Black cumin seeds than GA3 and
KNO3.
Keywords: Black cumin, Dormancy, germination, seed.
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INTRODUCTION
Black cumin (Nigella sativa L.) is an annual herbaceous plant belonging to the Ranunculaceae family [7] which
grows in countries around the Mediterranean Sea [7] as well as in other countries such as Iran. Seeds are angular, of
generally small size (1-5 mg), grey or black color. Possessing health-promoting benefits, black cumin seed is among
widely used seeds and has been employed either as a medicinal grain (in traditional medicine) or as a food
ingredient in several countries, including Egypt, Syria, Jordan and Iran [7]. Seeds of this plant have dormancy. Seed
dormancy has been described as “one of the least understood phenomena in seed biology” [5] and remains confusing
despite much recent progress. This confusion reflects the likelihood that dormancy is not a single phenomenon but a
condition with many contributing causes [6]. Traditionally this condition has been primarily negatively defined as a
developmental state in which a viable seed fails to germinate under superficially favorable environmental conditions
(e.g., adequate moisture). All types of dormancy impose a delay between seed shedding and germination, but the
underlying causes may vary. This variety has been classified in terms of whether germination is inhibited owing to
embryonic immaturity or physical or physiological constraints, and whether the controlling structure or substances
are embryonic or in the surrounding tissues of the seed, i.e., coat imposed [5]. Seed dormancy has been further
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negatively categorized in terms of the requirements for release from this block, such as disruption of the seed coat
(scarification), a period of dry storage (after-ripening) or moist chilling (stratification), or exposure to light [6]. The
situation is further complicated by the fact that, although germination is an all or nothing event for each seed,
populations display variable degrees of dormancy that are reflected in the rate or percentage of germination under
specific conditions. Presumably, each seed is in a state somewhere along the continuum from deeply dormant to
nondormant, but it remains unclear how the tipping point between nonpermissive and permissive for germination is
sensed for each seed [6]. However, this point is of critical agronomic and ecological significance because it
determines both the degree of synchronous germination in a given season and the reservoir of ungerminated viable
seeds remaining in the soil until a later season, i.e., the seed bank. Gibberellins [e.g., gibberellic acid (GA)] are a
family of 136 tetracyclic diterpenes, a small subset of which are active as plant hormones and known to stimulate
seed germination in a wide range of plant species; the predominant active GA depends on the species [16]. GA may
stimulate germination via the transition from embryonic to vegetative development, in part mediated by the
chromatin remodeling factor PICKLE (PKL) [8]. Incubation of seeds in moist conditions to break dormancy, usually
in cold to simulate overwintering is known as stratification [6]. The effect of GA3 as a germination promoter is
hypothesized to increase with stratification treatment [17]. Stratification also plays an important role in providing
the stimulus required to overcome dormancy. Stratification has been reported to induce an increase in GA3
concentration [2,17]. Many nitrogen-containing compounds, including NO gas, nitrite (NO2 −), nitrate (NO3 −),
nitrogen dioxide, ammonium, azide, and cyanide, promote dormancy release and seed germination in many species,
possibly as a means of sensing soil N availability [1]. Potassium nitrate is well documented as a compound, which
increases the germination of photo-dormant seeds [15]. Many gardeners choose potassium nitrate to break seed
dormancy and increase the health of plants [6]. The objective of this research was to determine the effects of
different seed dormancy breaking treatments which are able to stimulate and enhance germination of this important
medicinal plant.
MATERIALS AND METHODS
This study was carried out at the Department of Agronomy and plant breeding, Faculty of Agriculture, University of
Bu-Ali Sina, Islamic Republic of Iran. Black cumin seeds were received from Isfahan. Measured traits were: final
germination percentage, mean germination time, seedling length, seedling dry weight and vigor index.
Seed treatments
For stratification treatment, Black cumin seeds were mixed in perlite medium and distilled water in vessels, then
transferred to a refrigerator for 1, 2 and 3 weeks at 5±1◦C. These vessels put into sealed plastic bags to avoid
moisture loss. After this time the seeds were rinsed with distilled water three times. For GA3 treatment, seeds were
put into flasks contained 250, 500, 750, 1000 and 1250ppm Gibberellin for 24 hours (flasks were shaken on orbital
shaker under dark condition). For KNO3 treatment, seeds were treated by 0, 0.1, 0.2 and 0.3% (v/v) KNO3 for 24
hours. In this case, flasks were shaken on orbital shaker in light condition. Scarification with sulfuric acid 98% (v/v)
was done in 2 periods (30 and 60 seconds). For hot water treatment, seeds were treated at 70°C and 80°C in a hot
water bath, for 5 and 10 minutes, then taken away from the heat source. The seeds were left in the water overnight
(ca.12 hr) while it gradually cooled down to room temperature.
Germination tests
Three replicates of 100 seeds were germinated on top of double layered papers [9] with 5 ml of water in 10cm Petri
dishes. These Petri dishes were put into sealed plastic bags to avoid moisture loss. Seeds were allowed to germinate
at 20±1◦C in the dark condition for 21 days. Germination was considered to have occurred when the radicles were
2mm long. Germination percentage was recorded every 24h for 21 days. Mean germination time (MGT) was
calculated by following equation [14].
Mean
fi: Germination
Day during Time
germination (MGT):
period (between 0 and 28 day).
ni: Number of germinated seeds per day
N: Sum of germinated seeds
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Statistical analysis
The statistical design was a completely randomized design. Three replications and 100 seeds per replicate were
used. Data for germination and abnormal germination percentage were subjected to arcsine transformation before
analysis of variance. Statistical analysis was carried out using SAS program. Mean comparison was performed with
Duncan’s test at the 5% level of significance.
RESULTS AND DISCUSSION
There were significant differences (p80% germination was also achieved after the 1 and 2 weeks (Table 1). Concentration of 1250 ppm GA3
and 0.3% KNO3 had better effect than other treatments but they were no stronger action on germination percentage
of Black cumin seeds than 3 weeks stratification. However 1250 ppm GA3 was not different significantly to 0.3%
KNO3 as shown with the same letter (Table 1). Submersing seeds in hot water at 70°C and 80°C for 5 and 10
minutes and scarification with sulfuric acid had no effect on germination percentage compared to the control (Table
1).
Mean germination time (MGT)
The best treatment for this trait was detected in the stratification treatment for 3 weeks (3.72) and after this
treatment, application of 1250 ppm GA3 and 0.3% KNO3 were positive effect respectively (Table 1). Same as
germination percentage, application of sulfuric acid and hot water had not any effect on mean germination time
(Table 1).
Seedling Length (SL)
Comparison of means showed that stratification treatment for 3 weeks in this trait reached to its maximum rate
(4.16). Concentration of 1250 ppm GA3 and 0.3% KNO3 had good effect on seedling length but after 3 weeks
stratification (Table 1).
Seedling Dry Weight (SDW)
Scarification effect (with acid and hot water) on seedling dry weight was zero but the effect of stratification, GA3
and KNO3 were significant (Table 1). The highest seedling dry weight (0.0025) was observed after stratification in 3
weeks. Application of 1250 ppm GA3 was not different significantly to stratification in 3 weeks as shown with the
same letter (Table 1). Similarly, 0.3% of KNO3, also did increase the seedling dry weight of black cumin but not
stronger than stratification in 3 weeks and application of 1250 ppm GA3 (Table 1).
Vigour Index (VI)
Because of this trait is combined from germination percentage and seedling length, maximum amount of vigour
index was detected in 3 weeks stratification (Table 1). Application of 1250 ppm GA3 and 0.3% of KNO3 were after
3 weeks stratification respectively (Table 1).
In our experiment, neither sulfuric acid (98%) nor treating with hot water had any effect on germination indexes
compared to control group (Table 1). El Siddig et al. (2001) found that scarification with 90% sulfuric acid
decreased fresh weight, MGT and E1st of Tamaridus indica seedling. With increasing the concentration of GA3
from 250 to 1250ppm, all germination indexes were improved (Table 1). Gibberellins stimulate germination by
inducing hydrolytic enzymes that weaken the barrier tissues such as the endosperm or seed coat, inducing
mobilization of seed storage reserves, and stimulating expansion of the embryo [13]. Dormant seeds which require
chilling, dry storage after ripening and light as a germination stimulator, are often treated with GA3 to overcome
their dormancy [10]. At lower concentrations of GA3, germination was dearth. It seems low concentrations of GA3
had no strong effect on breaking dormancy. Levels of KNO3 did stimulate the germination of black cumin (Table
1). Shanmugavalli et al (2007) showed that seeds of sorghum soaked in 0.5% and 1% potassium nitrate (KNO3)
improved germination up to 44%. Potassium nitrate has been used for many years, with positive studies beginning in
the 1980's and it often increased the germination of photo-dormant [6]. According to Bewley and Black (1983),
KNO3 raises the ambient oxygen levels by making less oxygen available for citric acid cycle [15]. In addition to
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KNO3 and GA3 treatments, seed treatment with stratification for 1, 2 and 3 weeks were significant and dormancy of
black cumin seeds released by these treatments. Stratification for 3 weeks was more effective than 1 and 2 weeks
(Fig 1).
Table 1. Effect of seed dormancy breaking treatments on germination traits of Black cumin, In each column means followed by the
same letter are not significantly different at the P < 0.05 level
FGP MGT SL SDW VI
Dormancy breaking treatments
(%) (seed/day) (cm) (gr)
Control - - - - -
Acid scarification 98% (v/v)
Sulfuric acid (30 sec) - - - - -
Sulfuric acid (60 sec) - - - - -
Scarification by hot water
70°C + 5 min - - - - -
70°C + 10 min - - - - -
80°C + 5 min - - - - -
80°C + 10 min - - - - -
Gibberellic acid
250 ppm 22 f 7.81 f 1.1 f 0.001 f 24.2 j
500 ppm 35 e 6.66 e 1.75 e 0.0014 d 61.25 i
750 ppm 49 d 5.75 d 2.5 d 0.0016 c 122.5 g
1000 ppm 63 c 4.71 c 3.1 c 0.0022 abc 195.3 e
1250 ppm 76 b 4.11 b 4.06 ab 0.0025 a 308.56 b
Potassium nitrate (KNO3 v/v)
0.1 54 d 5.72 d 1.73 e 0.0011 e 93.42 h
0.2 64 c 4.66 c 2.44 d 0.0014 d 156.16 f
0.3 76.6 b 4.12 b 3.5 b 0.0023 ab 268.31 c
Stratification
1 week 65 c 5.7 d 2.48 d 0.0019 b 161.2 f
2 weeks 76 b 4.81 c 3c 0.0021 abc 228 d
3 weeks 82 a 3.72 a 4.16 a 0.0025 a 341.12 a
Dash in front of each combined treatment means zero germination
FGP: Final Germination Percentage, MGT: Mean Germination Time, SL: Seedling Length, SDW: Seedling Dry Weight, VI: Vigor
index
CONCLUSION
Fig 1. Comparison with stratification treatment for 3
weeks (A) and nontreated seeds (B) in Black cumin
(Nigella sativa L.)
Stratification plays an important role in improving sensitivity to other treatment to overcome dormancy [2, 13, 17].
Stratification has been reported to induce an increase in sensitivity to GA3 concentration [11]. Moreover, there are
various reports about the different physiological effects of gibberellings. For example, GA7 was more effective than
GA3 in promoting germination of Sanguinaria candensis L. (bloodroot) [3]. Finkelstein et al. (2008) mentioned that
GA may not trigger the onset of afterripening, but it may be necessary though not sufficient for seed dormancy
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release and germination. In this study 3 weeks of stratification, 1250ppm GA3 and 0.3% KNO3 were the most
effective treatment respectively. Similar to our results, Oh et al. (2006) suggest that stratification promotes
germination by increasing the potential for bioactive GA accumulation. Stratification led to increased expression of
the GA biosynthesis genes GA20ox1 (GIBBERELLIN 20 OXIDASE ), GA20ox2, and GA3ox1 and decreased
expression of the GA catabolic gene GA2ox2 (Yamauchi et al., 2004). In some reports stratification was introduced
as the best method for dormancy breaking. Beginning of the embryo dormancy is associated with accumulation of
growth inhibitors such as ABA and breaking of dormancy with a shift in the balance of growth regulators towards
growth promoters such as GA3, that overcome the effect of growth inhibitors [12].
The dormancy of Black cumin seed was broken by stratification, GA3 and KNO3treatment, but scarification with
hot water and sulfuric acid had no effect. Our results showed that applying stratification for 3 weeks could be the
best treatment for Black cumin dormant seeds (Fig 1). Finally, we think that Black cumin has morphophysiological
dormancy because it has underdeveloped embryo, but in addition it has a physiological component to their
dormancy. Therefore seeds require a dormancy-breaking treatment, for example a defined cold stratification which
in some cases can be replaced by GA application.
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