Journal of Forestry Research (2012) 23(4): 707−710 DOI 10.1007/s11676-012-0314-9
SHORT COMMUNICATION
Overcoming seed dormancy of mooseer (Allium hirtifolium) through cold stratification, gibberellic acid, and acid scarification Farshad Dashti • Hojat Ghahremani- Majd • Mahmood Esna-Ashari
Received: 2011-12-18
Accepted: 2012- 02-02
© Northeast Forestry University and Springer-Verlag Berlin Heidelberg 2012
Abstract : Mooseer (Allium hirtifolium Boiss.) is bulbous perennial herb widely used in pharmaceutical and food industry in Iran. We studied germination of mooseer seeds in two separate experiments. In the first experiment, we evaluated four treatments: sulfuric acid scarification, sandpaper scarification, cold stratification, and gibberellic acid (GA3) application. In the second experiment, we evaluated combinations of these treatments. All treatments in the first experiment had no effect on seed germination, suggesting that mooseer seeds have physical and physiological dormancy. In the second experiment, the highest germination percentage (86.6%) was observed after five minutes scarification with sulfuric acid (75% v/v), followed by 60 days of cold stratification. Duration of sulfuric acid scarification (5, 10, and 20 min) did not affect germination rates, but increasing duration of cold stratification (from 15 to 60 days), increased germination from 28.3% to 86.6%. Our study showed that mooseer seeds have both physical and physiological dormancy. Keywords: physical dormancy; physiological dormancy; germination; sulfuric acid; sand paper; Allium hirtifolium
Introduction Mooseer (Allium hirtifolium Boiss. synonym: A. stipitatum Regel.) is a bulbous perennial herb of the Alliaceae family, native to Iran and endemic in regions with elevations above 2000 m, mainly in the Zagross cold mountains in western Iran (Ebrahimi et al. 2009). Due to its antimicrobial (Taran et al. 2006) and antitumor (Ghodrati Azadi et al. 2008) properties, and its valuable food components (Ebrahimi et al. 2009), mooseer is widely used The online version is available at http:// www.springerlink.com Farshad Dashti (
)• Hojat Ghahremani- Majd • Mahmood Esna-Ashari
Department of Horticultural Sciences, Faculty of Agriculture, Bu-Ali Sina University, Hamedan 6517833131, Iran. E-mail:
[email protected];
[email protected] Responsible editor: Zhu Hong
in the pharmaceutical and food industries. Because of increasing mooseer consumption, it is necessary to produce this herb as a vegetable crop by developing methods for its propagation. Mooseer cloves and seeds can be used in propagation, but the cloves are very slow to grow and the seeds undergo deep dormancy. Neither is commercially efficient and therefore not recommended for large-scale propagation. Ecologically, seed dormancy is quite important in the wild, as it is an adaptive strategy that allows the seeds to retain viability during adverse environment conditions. This ensures that germination occurs at the appropriate time and seedlings are well-established and can survive in their natural habitats (Wang et al. 2007). However, dormant seeds require treatments before sowing. Generally, pre-sowing treatments such as cold stratification (Vandelook et al. 2009; Zhou et al. 2009), chemical or mechanical scarification (Chisha-kasumu et al. 2007; Olmez et al. 2007; Patane and Gresta 2006; Veassey and Teixeira De freitas 2002; Wang et al. 2007) and GA3 application (Nadjafi et al. 2006; Tigabu and Oden 2001) have been used to reduce seed hardness and to improve germination, emergence rate, and uniformity. Seed dormancy could therefore be an important limiting factor for Mooseer propagation. A successful method of breaking seed dormancy has not yet been reported. The aims of this study were to identify the type(s) of seed dormancy in mooseer and to develop methods for breaking seed dormancy.
Materials and methods Seeds were provided by the Agricultural and Natural Resources Research Center in Hamedan (34°36′ N, 48°20′ E). To segregate empty from whole seeds, all seeds were bathed in water and floating seeds were removed from the water surface. Seeds were then sterilized by soaking in 2% NaOCl solution for 5 min., followed by rinsing three times with distilled water prior to applying any treatment. All germination experiments were conducted using completely randomized design with three replications and each treatment contained 20 seeds. Seeds were placed on double
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Journal of Forestry Research (2012) 23(4): 707−710
filter paper discs (Whatman No.1) in 9-cm glass Petri dishes, moistened with 5-mL sterilized distilled water and incubated in a germinator at 20°C for 20 days. We carried out two separate experiments. In the first experiment, four treatments were separately applied to the seeds as follows: (1). Chemical scarification: seeds were treated with sulfuric acid (75% v/v) for 5, 10, 20, 40, 60, 80 and 100 min and then washed thoroughly with distilled water. (2). Cold stratification: seeds were kept at 4ºC in moistened peat for 15, 30, 45, 60, 90 and 120 days. (3). GA3 application: seeds were soaked in 0/025, 0/05, 0/1, 0/2, 0/5 and 1 mg.L-1 GA3 for 24 h. (4). Mechanical scarification: seeds were scarified with sand-paper. None of the above treatments had any effect on breaking dormancy and initiating seed germination. The second experiment was therefore conducted using combinations of treatments as follows: (1). Chemical scarification plus GA3 treatment: After chemical scarification with sulfuric acid (75% v/v) for 5, 10 and 20 min, seeds were soaked in GA3 (0/1, 0/2, 0/5 and 1 mg.L-1) for 24h. (2). Cold stratification and scarification with sulfuric acid and sand paper. Seeds were scarified with sand-paper and then by sulfuric acid (75% v/v) for 10 min, afterwards incubated at 4ºC for 30 days. (3). Chemical scarification plus cold stratification. Seeds were scarified with sulfuric acid (75% v/v) for 5, 10 and 20 min then incubated at 4ºC for 15, 30, 45 and 60 days. Germinated seeds were counted every day. Germination percentage (GP) and germination rate (GR) were calculated according to the following formula (Olmez et al. 2007). GP = n/v × 100
(1)
Where, n is the number of germinated seeds and v is the number of viable seeds initiated. GR = [(n1× t1) + (n2 × t2) + (ni × ti)] / T
(2)
where, ni is the number of days for each counting, ti is the number of germinated seeds in each counting, and T is the total number of germinated seeds. After Arcsine transformation, data were analyzed with SAS software. Mean comparisons were performed with Duncan’s multiple range test at the 1% level of significance.
ble seed dormancy. Table 1. Effects of GA3 and scarification with sulfuric acid on mooseer seed germination GA3 concentration
Soaking duration in
(mg⋅L-1)
sulfuric acid (min)
1000
500
200
100
GR (d)
20
30.00 az
4.20 e
10
23.00 b
4.70 cde 4.70 cde
5
21.60 bc
20
21.60 bc
4.40 de
10
18.30 bc
5.00 cd 5.00 cd
5
16.60 c
20
11.30 d
5.00 cd
10
8.30 de
5.30 bc
5
5.0 e
5.60 ab
20
6.60 e
5.60 ab
10
6.60 e
6.10 a
5
5.0 e
5.60 ab
0.00 g
0.00 g
Control
Notes: The similar letters in each column are not significantly different using Duncan’s multiple range tests at 1% level. GP is Germination percentage and GR is germination rate.
In the second treatment of the second experiment, sand paper and sulfuric acid scarification plus cold stratification for 30 days did not improve seed germination when applied separately, but the highest germination percentage (53%) was observed when sulfuric acid was combined with cold. However, the result of this combination did not differ significantly from that obtained by sand paper scarification plus cold stratification (Table 2). The results of this step proved that the seeds had double dormancy and required both scarification and cold stratification. The experiment was therefore finalized through the third treatment. Table 2. Effects of cold stratification and scarification with sulfuric acid and sand paper on mooseer seed germination Cold stratification (days)
Results All treatments of the first experiment had no effect on breaking mooseer seed dormancy (data not shown), suggesting that the seeds had double dormancy, so the second experiment was designed. From the first experiment it was clear that sulfuric acid scarification and GA3 treatments had no effect on seed germination when applied separately, but at the first stage of the second experiment the highest germination percentage (30%) and germination rate (4.2 days) were observed in the combination of sulfuric acid scarification for 20 min and the application of 1 mg·L-1 GA3 (Table 1). However this combination could not completely overcome the seed dormancy. This result suggested dou-
GP (%)
Scarification
GP (%)
GR
30
Sand paper
50 az
4.13 a
30
Sulfuric acid for 10 min
53 a
3.64 a
30
Without scarification
3b
5.66 a
0
Sand paper
0c
0.00 b
0
Sulfuric acid for 10 min
0c
0.00 b
Notes: Data represent means of three replicates compared by Duncan's multiple range test (p< 0.01). GP is Germination percentage and GR is germination rate.
The highest germination percentage (86.6%) was obtained from scarification by sulfuric acid (75% v/v) for 5 min plus cold stratification for 60 days (Table 3). This combination produced results similar to those after 10 or 20 min sulfuric acid treatment plus 60 days cold stratification: both combinations showed more than 80% germination percentage. The results were the same for
Journal of Forestry Research (2012) 23(4): 707−710
709
germination rate. By increasing the duration of cold stratification from 15 to 60 days, both germination percentage and rate were increased. In order to attain an acceptable number of germinated seeds, both scarification and cold stratification (at least 60 days) were needed. Duration of soaking in sulfuric acid did not affect germination. Table 3. Effects of cold stratification and scarification with sulfuric acid on mooseer seed germination Cold stratifi-
Soaking duration in
cation (days)
sulfuric acid (min)
15
30
45
60
Control
GP (%)
GR
5
30.00 ez
6.09 a
10
28.30 e
5.82 a
20
28.30 e
5.27 b
5
40.00 d
4.87 b
10
53.30 d
3.82 c
20
48.30 d
3.79 c
5
68.30 c
3.51 cd
10
73.30 bc
3.54 cd
20
70.00 bc
3.47 cd
5
86.60 a
3.09 e
10
81.60 ab
3.28 de
20
80.00 ab
3.15 de
0.00 f
0.00 f
Notes: Data represent means of three replicates compared by Duncan's multiple range test (p< 0.01). GP is Germination percentage and GR is germination rate.
Discussion None of the treatments affected mooseer seed germination when applied separately. In contrast, the combination of scarification and stratification was successful in breaking dormancy and improving seed germination. This suggested that seeds of mooseer had double dormancy. The highest germination value (GP and GR) was obtained from cold stratification at 4ºC following scarification with sulfuric acid or sand paper. This indicated that the seed coat and embryo were the two factors limiting mooseer seed germination. Germination value of seeds was significantly affected by scarification. Without scarification, none of the seeds germinated even after 120 days of cold stratification. In contrast, when seeds were soaked in sulfuric acid before cold stratification, the germination percentage increased to 86.6%, indicating that the hard seed coat acted as a physical barrier to embryo emergence. Immersion of the seeds for more than 5 min in sulfuric acid reduced their germination, probably because of harmful effects of the acid on embryos. The use of sulfuric acid as a scarification agent to overcome seed coat dormancy is well known and several studies have shown that sulfuric acid scarification can improve seed germination (De Villiers et al. 2002; Hermansen et al. 2000; Tigabu and Oden 2001; Veassey and Teixeira De freitas 2002). Sulfuric acid treatment damages the surface of the seed coat or splits the palisade layer of the micropylar, thereby facilitating the
diffusion of oxygen and water and reducing mechanical resistance to radical emergence (Schelin et al. 2003). A hard seed coat provides important ecological advantages to many wild species. This factor favors the persistence of seeds in the soil and increases their vigor by enhancing germination. Cold stratification significantly increased germination values when applied in combination with sulfuric acid or sand paper scarification. Cold stratification has been applied to dormant seeds of many species and has been reported as a successful method for alleviating endogenous dormancy (Li et al. 2007). This study proved that cold stratification had the greatest effect in enhancing mooseer seed germination. The prolonged cold stratification required to break mooseer seed dormancy supported the hypothesis that strong dormancy resides in the embryo of this herb. As mooseer is grown in cold mountains, embryo dormancy can be an ecological strategy to stop germination until the next spring after the seeds have been cold-stratified during winter. The results obtained from sand paper and sulfuric acid scarification showed that both mechanical and chemical scarification aided breakdown of seed dormancy and enabled germination. The positive effect of mechanical scarification on seed germination is common in many hard-seeded plant species (Malavasi and Malavasi 2004; Ortega Baes et al. 2002; Patane and Gresta 2006; Travlos et al. 2007). Exogenously applied GA3 had a little effect on breaking the dormancy of mooseer seeds. The treatment of high concentration of GA3 (1%) and scarification with sulfuric acid for 20 min increased the germination up to 30%, compared with the control. The actual mechanism of GA3 in breaking seed dormancy is not understood (Chisha-kasumu et al. 2007). GA3 application enhances seed germination in some plant species (Chuanren et al. 2004; Nadjafi et al. 2006; Tigabu and Oden 2001), but these studies have shown that exogenous application of GA3 can show different effects on seed germination among plant species (Schelin et al. 2003). The limited effect of GA3 might be due to the fact that it cannot replace cold stratification in Mooseer seeds because GA3 does not overcome the requirement for chilling. Other reports show that GA3 has no effect on breaking seed dormancy (Baskin et al. 2000; Hidayati et al. 2001; Schelin et al. 2003; Zhou et al. 2009).
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