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Hardseedness of Ammopiptanthus nanus and Ammopiptanthus mongolicus
ARTICLE IN PRESS Journal of Arid Environments Journal of Arid Environments 72 (2008) 263–269 www.elsevier.com/locate/jaridenv
Short communication
Hardseedness of Ammopiptanthus nanus and Ammopiptanthus mongolicus Q.-H. Yanga,b, W.-H. Yea,, X.-J. Gea, X.-J. Yinb a
South China Botanical Garden, The Chinese Academy of Sciences, Guangzhou 510650, People’s Republic of China b Jiaying University, Meizhou 514015, People’s Republic of China Received 26 October 2006; received in revised form 10 May 2007; accepted 10 May 2007 Available online 29 June 2007
Abstract Hardseedness of Ammopiptanthus mongolicus and Ammopiptanthus nanus was examined through imbibition, germination, and ageing testing in this study. The effects of soaking duration, soaking temperature, hardseedness level, and seed coat color on seed imbibition were investigated. Germinability and viability of hard seeds and soft seeds (used as controls) were also analyzed through ageing testing. Imbibition percentage increased with lengthening of soaking duration, rising of soaking temperature, decreasing of seed moisture content, and darkening of seed coat color. Harder seeds had higher germination percentage and germination index compared to softer seeds. They tolerated ageing and their storability was higher. The seeds of both plants tended to become hard at maturity with long storability, which could be considered as an adaptation to the dry and extremely cold environments in the desert of Northwest China. However, hard seeds could not imbibe and germinate. r 2007 Elsevier Ltd. All rights reserved. Keywords: Ageing; Germination; Imbibition; Seed
1. Introduction Ammopiptanthus Cheng f. (Fabaceae) is the sole genus of evergreen broadleaf shrubs in the desert of China, and comprises of only two species: Ammopiptanthus mongolicus and Ammopiptanthus nanus (Cheng, 1959; Pan and Huang, 1993). Both species are narrowly distributed; A. mongolicus is endemic to the south Gobi desert, and A. nanus is restricted to the borders between China and Kyrgyzstan, growing in a narrow altitudinal strip between 1800 and 2800 m (Pan et al., 1992). Natural regeneration of both species is limited because of low seed germination (Liu, 1998; Pan et al., 1992; Yang et al., 2004). Moreover, in the past three decades, both species have rapidly declined, mainly due to increasing anthropogenic pressures in their natural areas. Thus, they have been categorized as ‘endangered’ and given protected status in China (Fu, 1989). It is known that Ammopiptanthus species are important as it reduces sand erosion and delays desertification. However, questions remain on germination and establishment seedlings. The major objective of this study was to investigate the hardseedness and its influence on seed imbibition. Corresponding author. Tel.: +86 137 10546160.
E-mail addresses: [email protected], [email protected] (Q.-H. Yang), [email protected] (W.-H. Ye). 0140-1963/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaridenv.2007.05.008
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2. Materials and methods All the seeds used in this study were harvested during autumn 2003. A. nanus seeds were collected in natural habitats in Wuqia of Xinjiang Uygur Autonomous Region in China and Turpan Eremophytes Botanic Garden of Xinjiang Institute of Biology (401510 N, 981110 E), the Chinese Academy of Sciences. A. mongolicus seeds were collected in Dengkou Psammophyte Botanical garden (Inner Mongolia) (491290 N, 1061350 E), the Chinese Academy of Forestry Sciences. The seeds were immediately taken out of the pods after harvesting, and then air-dried for 3 days at ambient temperature before carrying out the experiments described below. 2.1. Seed imbibition tests In all imbibition tests, three samples of 200 seeds each were soaked in 120 mm diameter Petri dishes with distilled water at ambient temperature (18–25 1C) for 90 h, and the numbers of imbibed seeds was recorded after soaking for 6, 18, 30, 42, 54, 78, 90 h. The imbibed seeds were those whose size increased at least two times their original size. Imbibition percentage was calculated using Imbibition percentage ð%Þ ¼
number of imbibed seeds 100. number of soaked seeds
2.1.1. Imbibition tests of seeds with different moisture contents Seeds with three moisture content levels (20%, 10%, and 8%) were used in imbibition tests. Seeds freshly harvested, air-dried at ambient temperature in airtight glass desiccators containing active silica gel for 3 days or dried for 7 days resulted in these moisture content levels, respectively. 2.1.2. Imbibition tests at different temperatures Seeds of both species with approximately 10% MC were soaked in water at 10 1C, ambient temperature, and 30 1C. 2.1.3. Imbibition tests of seeds with various colors Yellow, yellowish black and black seeds of A. nanus, and yellow, yellowish brown and black seeds of A. mongolicus with about 10% moisture content were soaked in water at ambient temperature. Color of seed coat darkens with increasing maturity. 2.1.4. Definition of hardseedness level The seeds were placed in Petri dishes with distilled water in growth chambers (PYX-300G-A; Shaoguan Keli Experimental Instrument Co. Ltd., Shaoguan, China) set at 30 1C. Hardseedness levels of seeds were determined by the length of time taken for the seeds to imbibe: the longer the imbibition duration, the higher the hardseedness. The imbibed seeds were counted and the IP was calculated for imbibition durations 12, 24, 36, 48, and 60 h (CK1, CK2, CK3, CK4, and CK5). The seeds that did not imbibe after soaking for 60 h were regarded as hard seeds (H). 2.2. Seed germination tests The fresh or imbibed seeds were soaked in distilled water for about 5 h, surface disinfected with 0.1% mercuric chloride solution for 10 min, rinsed five times in sterile distilled water, and germinated for 10 days in growth chambers at constant 25 1C with 12 h daily light at 2000–3000 lx provided by cool white fluorescent lamps. Three samples, 50 seeds of each seed hardseedness level and imbibition treatment were placed in Petri dishes with 0.8–1% agar, one sample per dish. The hard seeds were soaked for 2 h in distilled water at 50–60 1C, and those that did not imbibe were further soaked in 15% H2O2 solution for 20 min before being used in germination tests. Germination was defined as the appearance of a radicle more than 0.5 cm in length,
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and germination percentage and germination index were calculated as follows: germination percentage ð%Þ ¼ germination index ¼
X Gt Dt
number of germinated seeds 100, number of total seeds per sample
,
where Gt means numbers of germinated seeds after t days. 2.3. Seed ageing tests The seeds were aged under constant temperature 45 1C and relative humidity 100% for 1, 3, 5, and 7 days. Then, they were soaked for 60 h in distilled water at ambient temperature. If the size of a seed increased in size to over two times after soaking, it was regarded as a soft seed; otherwise, it was considered a hard seed. The germination, leakage rate of electrolyte,, and dehydrogenase activity were measured after ageing. 2.3.1. Determination of leakage rate of electrolyte Three samples of 20 seeds each per ageing treatment were soaked in 50 ml double-distilled water for 24 h at ambient temperature, boiled at 100 1C for 30 min and then cooled down at ambient temperature. The leachate conductivity was determined by an electrical conductivity determinator (DDS-11A; Shanghai No.2 Analytical Instrument Co., China) at 3072 1C. Relative electric conductivity ¼ electric conductivity before boiling/ electric conductivity after boiling. 2.3.2. Determination of dehydrogenase activity Dehydrogenase activity was determined by the quantitative test of triphenyl tetrazolium chloride (TTC). Three samples of 20 each per ageing treatment were germinated for 24 h in an incubator at constant 25 1C with 12 h light at 2000–3000 lx. The hard seeds were scraped, then immersed in 20 ml 0.1% TTC solution for 3 h, and then rinsed three times with distilled water. The rinsed seeds were immersed in 20 ml 95% alcohol solution in sealed test tubes put in an oven at 30 1C. Optical density of the solution was measured with a UV–vis spectrophotometer (Cambds UV/vis; Perkin Elmer Instrument, USA) at a wavelength of 490 nm. 2.4. Data analysis The germination percentage, imbibition percentage, and relative electric conductivity were transformed into arcsine values and subjected to ANOVA using SPSS (11.0) package to test if there were significant differences among treatments, and LSD test was used to determine if there were significant differences between treatments. 3. Results 3.1. Effect of seed moisture content and temperature on imbibition percentage As soaking time increases, the imbibition percentage rises more rapidly in seeds with higher moisture content, than with lower moisture content (Fig. 1). The imbibition percentage of A. nanus and A. mongolicus seeds with 20% moisture content was 100% after 6 h of soaking. With 10%, the imbibition percentage of A. nanus seeds reached 36.67% and 94.67%, after imbibition for 6 and 90 h, respectively, and for A. mongolicus seeds the corresponding figures were 39.66% and 95.67%. However, the seed imbibition of both species with 8% moisture content reached only 12% and 70.33% after 6 and 90 h, respectively. The imbibition percentage of seeds soaked at higher temperature rises more quickly over time (Fig. 1). After soaking for 42 h, the imbibition percentage of seeds of both species reached 100% at 30 1C and reached 83.33% at ambient temperature. But at 10 1C it reached only 43.67% for A. nanus and 59.33% for A. mongolicus seeds.
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80
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60
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40
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20
0
0 0
15
30
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90
0
15
Duration of soaking (h) MC8%
MC10%
30
45
60
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90
Duration of soaking (h) MC20%
MC8%
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100
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80
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60
40
MC10%
MC20%
40 A.nanus
20
20
0
A. nanus
0 0
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75
Duration of soaking (h) 10 °C 30 °C AT
90
0
15
30
45
60
75
90
Duration of soaking (h) 10 °C
AT
30 °C
Fig. 1. Effect of seed MC and soaking duration on seed imbibition percentage (MC means moisture content, AT means ambient temperature (po0.05, Fisher’s LSD test).
3.2. Effect of seed coat color on imbibition percentage The imbibition percentage of seeds with different seed coat colors of both species rose with increase in soaking duration. After soaking for 90 h, the imbibition percentages of A. nanus seeds with yellow, yellowishblack, and black seed coat were 51.67%, 41.33%, and 34.33%, respectively, and those of A. mongolicus seeds with yellow, yellowish-brown, and brown seed coat were 76.33%, 56.00%, and 47.33%, respectively. 3.3. Hardseedness and germination capacity The germination percentage and germination index were not significantly different between hard seeds (H) and seeds of CK5, but they were significantly larger for hard seeds than for seeds of CK1 and CK2. The germination percentage of hard seeds of both species was nearly 20% more than that of CK1 seeds, but there was no significant difference between germination percentage of CK3, CK4, and CK5 seeds. After soaking in water at 50–60 1C and H2O2 solution, germination percentage and germination index of hard seeds were more than those of other seeds. 3.4. Seed ageing The germination percentage, germination index, and dehydrogenase activity of soft seeds decreased, and relative electric conductivity increased as ageing time increased; those of hard seeds did not have significant change after ageing for 1 day, but all changed significantly after 3 and more days (Table 1). There was a significantly negative relationship between germination index and days of ageing, but a positive relationship between the electrical conductivity rate and days of ageing for soft seeds (po0.05). The germination
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Table 1 Changes of GP, GI, REC and DA of Ammopiptanthus seeds after ageing for various days Species name
Ageing days
GP
GI
DA (OD490)
REC (%)
Hard seed
Control
Hard seed
Control
Hard seed
Control
Hard seed
Control
A. nanus
0 1 3 5 7
94.50 96.00 94.00 86.00 71.50
a a ab b c
85.00 78.50 71.50 62.50 48.00
a a b c d
24.23 25.17 24.69 23.15 20.43
a a a ab b
23.38 20.73 17.44 15.50 12.31
a a b bc c
42.33 44.67 51.67 60.33 66.33
a a b c c
47.33 57.67 67.33 78.00 84.00
a b c d d
0.46 0.42 0.38 0.36 0.30
a ab b b c
0.44 0.35 0.27 0.22 0.17
a b c cd d
A. mongolicus
0 1 3 5 7
90.50 88.50 87.00 77.50 70.50
a a ab bc c
85.00 84.50 73.00 64.00 45.50
a a b c d
23.87 23.25 22.32 20.78 20.13
a a a ab b
23.38 22.89 20.71 18.75 15.56
a a a ab b
48.00 51.33 62.00 65.00 71.33
a a b b c
51.00 64.00 77.67 79.00 86.33
a b c c
0.45 0.43 0.37 0.34 0.31
a a b bc c
0.46 0.37 0.32 0.24 0.18
a b bc d d
Note: GP, GI, REC, and DA means germination index, germination percentage, relative electrical conductivity, and dehydrogenase activity, respectively. The values with the same letter in the same column had no significant difference (po0.05, Fisher’s LSD test). The control was the soft seeds.
percentage, germination index, and dehydrogenase activity of hard seeds decreased and the electrical conductivity rate increased more slowly than those of soft seeds as ageing duration increased (Table 1). 4. Discussion and conclusions Our tests indicate that seed imbibition percentage of A. nanus and A. mongolicus decreased as seed moisture content and imbibition temperature decreased. The high hardseedness prevents immediate germination of Ammopiptanthus seeds in dry seasons since seeds do require a minimum soil moisture content to imbibe. We observed that imbibition percentage was lower in dark colored seeds of A. nanus and A. mongolicus. In other plant species, seed coat color darkens with increasing seed maturity (Argel and Humphryes, 1983), and seeds with unpigmented seed coat may deteriorate more rapidly and can be more susceptible to imbibition damage than those with pigmented seed coat (Abdullah et al., 1991; Asiedu and Powell, 1998). The present study also showed that as the seed coat darkened, the seed imbibition percentage decreased. Other researches have shown that soybean seeds with black seed coat had slower initial imbibition rates, higher resistance to field deterioration, and thicker and tougher seed coat than those with non-black seed coat (Chachalis and Smith, 2000; Krzyzanowski et al., 1999). Seed coats of plant species in 15 families, including Fabaceae, are temporarily impermeable to water and gases and do not germinate even if they are in conditions ideal for germination (Souza and Marcos-Filho, 2001). High hardseedness is the reason for seed coat-imposed dormancy (Bewley and Black, 1994), or physical dormancy (Baskin et al., 2000). Our ageing test using electrical conductivity of electrolytes leached from imbibed seeds and dehydrogenase activity of TTC solutions showed the hard seeds A. nanus and A. mongolicus can tolerate ageing and thus adverse storage conditions better than soft seeds. So the hard seeds had better storability than soft ones. There was hardseedness variation within seed population of A. nanus and A. mongolicus and the germination varied with hardseedness level (Fig. 2). Germination percentage and germination index of seeds rose gradually with increase in hardseedness level, but the hard seeds required pretreatments to relieve the constraint of seed coat before germination. The hardseedness variation with moisture content, seed coat color, and soaking duration could be necessary for the populations to persist in environments that change in both time and space, and could be a survival strategy to ensure that germination occurs under the most favorable environments. For germplasm conservation, the seeds with higher hardseedness should be preferred to those with lower hardseedness. In arid and semi-arid areas, many legume species have delayed hard-seed breakdown patterns with most seeds remaining hard until late autumn (Norman et al., 1998). Buried seeds of some legume species can survive
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30
100 80
ab
a
a
a
a
25
b
a
a
a
b
20 GI
60
GP(%)
ab c
40
15 10
20
5
0
0 CK1
CK2
CK3
CK4
CK5
H
CK1
CK2
CK3
CK4
CK5
H
Hardseedness level
Hardseedness level A. nanus 30
100
bc e
60
a
ab
25
d
20 f
GI
GP(%)
80
ab
cd
a
a
bc
d
15
40
10
20
5
0 CK1
CK2
CK3 CK4 Hardseedness level
CK5
H
CK1
CK2
CK3
CK4
CK5
H
Hardseedness level A. mongolicus
Fig. 2. Germination capability of seeds with various hardseedness levels. Values with the same letters on left corner are not significantly different at p ¼ 0.05 in Fisher’s LSD test.
as hard seeds for up to 12 years without significant loss of viability (Taylor and Ewing, 1988), which increases the seed longevity and population persistence. High hardseedness contributes to persistent soil seed bank by preventing germination from season rainfall and allows a certain proportion of a seed population to survive even in extended drought (Norman et al., 1998). It is also one of the most important adaptive and survival strategy of Ammopiptanthus species. The extended germination period caused by high hardseedness could improve the chances to get a good stand, since the hardseedness varies and this determines seed dormancy or readiness to germinate. Acknowledgments We thank Dr. Wang Zhangming and two anonymous referees for advice on data analysis and language revision. This research was funded by State Key Basic Research and Development Plan of China (G2000046803) and Key Natural Scientific Projects of Guangdong Province, China (021536). References Abdullah, W.D., Powell, A.A., Matthews, S., 1991. Association of differences in seed vigour in long bean (Vigna sesquipedalis) with testa colour and imbibition damage. Journal of Agricultural Science 116, 259–264. Argel, P.J., Humphryes, L.R., 1983. Environment effects on the seed development and the hardseededness in Stylosanthes hamata cv. Verano. I. Temperature. Australian Journal of Agricultural Research 34, 261–270. Asiedu, E.A., Powell, A.A., 1998. Comparisons of the storage potential of cultivars of cowpea (Vigna unguiculata) differing in seed coat pigmentation. Seed Science and Technology 26, 211–221. Baskin, J.M., Baskin, C.C., Li, X., 2000. Taxonomy, anatomy and evolution of physical dormancy. Plant Species Biology 15, 139–152. Bewley, J.D., Black, M., 1994. Seeds: Physiology of Development and Germination. Plenum Press, New York, pp. 147–197. Chachalis, D., Smith, M.L., 2000. Imbibition behavior of soybean (Glycine max (L.) Merril) accessions with different testa characteristics. Seed Science and Technology 28, 321–331. Cheng, S.H., 1959. Ammopiptanthus Cheng f. A new genus of Leguminosae from central Asia. Journal of Botany (USSR) 44, 1381–1386.
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Fu, L.G., 1989. The Rare and Endangered Plants in China. Shanghai Education Press, Shanghai. Krzyzanowski, F.C., Franc- aneto, J.B., Kaster, M., Mandarino, J.M.G., 1999. Metodologia para selec- a˜o de geno´tipos de soja com semente resistente ao dano mecaˆnico—relac- a˜o com o conteu´do de lignina. In: Resultados de pesquisa da Embrapa Soja, 1998. Londrina, EMBRAPA/CNPSo, pp. 213–214. Liu, G.H., 1998. Study on the endangered reasons of Ammopiptanthus mongolicus in the desert of Alashan. Bulletin Botanical Research 18, 341–345. Norman, H.C., Cocks, P.S., Smith, F.P., Nutt, B.J., 1998. Reproductive strategies in Mediterranean annual clovers: germination and hardseededness. Australian Journal of Agricultural Research 49, 973–982. Pan, B.R., Huang, S.P., 1993. Cytological study of the genus Ammopipthanthus. Acta Botanica Sinica 35, 314–317. Pan, B.R., Yu, Q.L., Yan, C., 1992. Study for the ecological environment and vulnerable reasons of the Ammopiptanthus nanus. Acta Phytecologia et Geobotanica Sinica 16, 276–282. Souza, F.H.D., Marcos-Filho, J., 2001. The seed coat as a modulator of seed–environment relationships in Fabaceae. Revista Brasileira de Botaˆnica 24, 365–375. Taylor, G.B., Ewing, M.A., 1988. Effect of depth of burial on the longevity of hard seeds of subterranean clover and annual medics. Australian Journal of Experimental Agriculture 28, 77–81. Yang, Q.H., Ge, X.J., Ye, W.H., Deng, X., Liao, F.L., 2004. Characteristics of Ammopiptanthus nanus seed and factors affecting its germination. Acta Phytoecologica Sinica 28, 651–656.
Short communication
Hardseedness of Ammopiptanthus nanus and Ammopiptanthus mongolicus Q.-H. Yanga,b, W.-H. Yea,, X.-J. Gea, X.-J. Yinb a
South China Botanical Garden, The Chinese Academy of Sciences, Guangzhou 510650, People’s Republic of China b Jiaying University, Meizhou 514015, People’s Republic of China Received 26 October 2006; received in revised form 10 May 2007; accepted 10 May 2007 Available online 29 June 2007
Abstract Hardseedness of Ammopiptanthus mongolicus and Ammopiptanthus nanus was examined through imbibition, germination, and ageing testing in this study. The effects of soaking duration, soaking temperature, hardseedness level, and seed coat color on seed imbibition were investigated. Germinability and viability of hard seeds and soft seeds (used as controls) were also analyzed through ageing testing. Imbibition percentage increased with lengthening of soaking duration, rising of soaking temperature, decreasing of seed moisture content, and darkening of seed coat color. Harder seeds had higher germination percentage and germination index compared to softer seeds. They tolerated ageing and their storability was higher. The seeds of both plants tended to become hard at maturity with long storability, which could be considered as an adaptation to the dry and extremely cold environments in the desert of Northwest China. However, hard seeds could not imbibe and germinate. r 2007 Elsevier Ltd. All rights reserved. Keywords: Ageing; Germination; Imbibition; Seed
1. Introduction Ammopiptanthus Cheng f. (Fabaceae) is the sole genus of evergreen broadleaf shrubs in the desert of China, and comprises of only two species: Ammopiptanthus mongolicus and Ammopiptanthus nanus (Cheng, 1959; Pan and Huang, 1993). Both species are narrowly distributed; A. mongolicus is endemic to the south Gobi desert, and A. nanus is restricted to the borders between China and Kyrgyzstan, growing in a narrow altitudinal strip between 1800 and 2800 m (Pan et al., 1992). Natural regeneration of both species is limited because of low seed germination (Liu, 1998; Pan et al., 1992; Yang et al., 2004). Moreover, in the past three decades, both species have rapidly declined, mainly due to increasing anthropogenic pressures in their natural areas. Thus, they have been categorized as ‘endangered’ and given protected status in China (Fu, 1989). It is known that Ammopiptanthus species are important as it reduces sand erosion and delays desertification. However, questions remain on germination and establishment seedlings. The major objective of this study was to investigate the hardseedness and its influence on seed imbibition. Corresponding author. Tel.: +86 137 10546160.
E-mail addresses: [email protected], [email protected] (Q.-H. Yang), [email protected] (W.-H. Ye). 0140-1963/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaridenv.2007.05.008
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2. Materials and methods All the seeds used in this study were harvested during autumn 2003. A. nanus seeds were collected in natural habitats in Wuqia of Xinjiang Uygur Autonomous Region in China and Turpan Eremophytes Botanic Garden of Xinjiang Institute of Biology (401510 N, 981110 E), the Chinese Academy of Sciences. A. mongolicus seeds were collected in Dengkou Psammophyte Botanical garden (Inner Mongolia) (491290 N, 1061350 E), the Chinese Academy of Forestry Sciences. The seeds were immediately taken out of the pods after harvesting, and then air-dried for 3 days at ambient temperature before carrying out the experiments described below. 2.1. Seed imbibition tests In all imbibition tests, three samples of 200 seeds each were soaked in 120 mm diameter Petri dishes with distilled water at ambient temperature (18–25 1C) for 90 h, and the numbers of imbibed seeds was recorded after soaking for 6, 18, 30, 42, 54, 78, 90 h. The imbibed seeds were those whose size increased at least two times their original size. Imbibition percentage was calculated using Imbibition percentage ð%Þ ¼
number of imbibed seeds 100. number of soaked seeds
2.1.1. Imbibition tests of seeds with different moisture contents Seeds with three moisture content levels (20%, 10%, and 8%) were used in imbibition tests. Seeds freshly harvested, air-dried at ambient temperature in airtight glass desiccators containing active silica gel for 3 days or dried for 7 days resulted in these moisture content levels, respectively. 2.1.2. Imbibition tests at different temperatures Seeds of both species with approximately 10% MC were soaked in water at 10 1C, ambient temperature, and 30 1C. 2.1.3. Imbibition tests of seeds with various colors Yellow, yellowish black and black seeds of A. nanus, and yellow, yellowish brown and black seeds of A. mongolicus with about 10% moisture content were soaked in water at ambient temperature. Color of seed coat darkens with increasing maturity. 2.1.4. Definition of hardseedness level The seeds were placed in Petri dishes with distilled water in growth chambers (PYX-300G-A; Shaoguan Keli Experimental Instrument Co. Ltd., Shaoguan, China) set at 30 1C. Hardseedness levels of seeds were determined by the length of time taken for the seeds to imbibe: the longer the imbibition duration, the higher the hardseedness. The imbibed seeds were counted and the IP was calculated for imbibition durations 12, 24, 36, 48, and 60 h (CK1, CK2, CK3, CK4, and CK5). The seeds that did not imbibe after soaking for 60 h were regarded as hard seeds (H). 2.2. Seed germination tests The fresh or imbibed seeds were soaked in distilled water for about 5 h, surface disinfected with 0.1% mercuric chloride solution for 10 min, rinsed five times in sterile distilled water, and germinated for 10 days in growth chambers at constant 25 1C with 12 h daily light at 2000–3000 lx provided by cool white fluorescent lamps. Three samples, 50 seeds of each seed hardseedness level and imbibition treatment were placed in Petri dishes with 0.8–1% agar, one sample per dish. The hard seeds were soaked for 2 h in distilled water at 50–60 1C, and those that did not imbibe were further soaked in 15% H2O2 solution for 20 min before being used in germination tests. Germination was defined as the appearance of a radicle more than 0.5 cm in length,
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and germination percentage and germination index were calculated as follows: germination percentage ð%Þ ¼ germination index ¼
X Gt Dt
number of germinated seeds 100, number of total seeds per sample
,
where Gt means numbers of germinated seeds after t days. 2.3. Seed ageing tests The seeds were aged under constant temperature 45 1C and relative humidity 100% for 1, 3, 5, and 7 days. Then, they were soaked for 60 h in distilled water at ambient temperature. If the size of a seed increased in size to over two times after soaking, it was regarded as a soft seed; otherwise, it was considered a hard seed. The germination, leakage rate of electrolyte,, and dehydrogenase activity were measured after ageing. 2.3.1. Determination of leakage rate of electrolyte Three samples of 20 seeds each per ageing treatment were soaked in 50 ml double-distilled water for 24 h at ambient temperature, boiled at 100 1C for 30 min and then cooled down at ambient temperature. The leachate conductivity was determined by an electrical conductivity determinator (DDS-11A; Shanghai No.2 Analytical Instrument Co., China) at 3072 1C. Relative electric conductivity ¼ electric conductivity before boiling/ electric conductivity after boiling. 2.3.2. Determination of dehydrogenase activity Dehydrogenase activity was determined by the quantitative test of triphenyl tetrazolium chloride (TTC). Three samples of 20 each per ageing treatment were germinated for 24 h in an incubator at constant 25 1C with 12 h light at 2000–3000 lx. The hard seeds were scraped, then immersed in 20 ml 0.1% TTC solution for 3 h, and then rinsed three times with distilled water. The rinsed seeds were immersed in 20 ml 95% alcohol solution in sealed test tubes put in an oven at 30 1C. Optical density of the solution was measured with a UV–vis spectrophotometer (Cambds UV/vis; Perkin Elmer Instrument, USA) at a wavelength of 490 nm. 2.4. Data analysis The germination percentage, imbibition percentage, and relative electric conductivity were transformed into arcsine values and subjected to ANOVA using SPSS (11.0) package to test if there were significant differences among treatments, and LSD test was used to determine if there were significant differences between treatments. 3. Results 3.1. Effect of seed moisture content and temperature on imbibition percentage As soaking time increases, the imbibition percentage rises more rapidly in seeds with higher moisture content, than with lower moisture content (Fig. 1). The imbibition percentage of A. nanus and A. mongolicus seeds with 20% moisture content was 100% after 6 h of soaking. With 10%, the imbibition percentage of A. nanus seeds reached 36.67% and 94.67%, after imbibition for 6 and 90 h, respectively, and for A. mongolicus seeds the corresponding figures were 39.66% and 95.67%. However, the seed imbibition of both species with 8% moisture content reached only 12% and 70.33% after 6 and 90 h, respectively. The imbibition percentage of seeds soaked at higher temperature rises more quickly over time (Fig. 1). After soaking for 42 h, the imbibition percentage of seeds of both species reached 100% at 30 1C and reached 83.33% at ambient temperature. But at 10 1C it reached only 43.67% for A. nanus and 59.33% for A. mongolicus seeds.
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Imbibition percentage (%)
266
100
100
80
80
60
60
40
40
20
20
0
0 0
15
30
45
60
75
90
0
15
Duration of soaking (h) MC8%
MC10%
30
45
60
75
90
Duration of soaking (h) MC20%
MC8%
100
100
80
80
60
60
40
MC10%
MC20%
40 A.nanus
20
20
0
A. nanus
0 0
15
30
45
60
75
Duration of soaking (h) 10 °C 30 °C AT
90
0
15
30
45
60
75
90
Duration of soaking (h) 10 °C
AT
30 °C
Fig. 1. Effect of seed MC and soaking duration on seed imbibition percentage (MC means moisture content, AT means ambient temperature (po0.05, Fisher’s LSD test).
3.2. Effect of seed coat color on imbibition percentage The imbibition percentage of seeds with different seed coat colors of both species rose with increase in soaking duration. After soaking for 90 h, the imbibition percentages of A. nanus seeds with yellow, yellowishblack, and black seed coat were 51.67%, 41.33%, and 34.33%, respectively, and those of A. mongolicus seeds with yellow, yellowish-brown, and brown seed coat were 76.33%, 56.00%, and 47.33%, respectively. 3.3. Hardseedness and germination capacity The germination percentage and germination index were not significantly different between hard seeds (H) and seeds of CK5, but they were significantly larger for hard seeds than for seeds of CK1 and CK2. The germination percentage of hard seeds of both species was nearly 20% more than that of CK1 seeds, but there was no significant difference between germination percentage of CK3, CK4, and CK5 seeds. After soaking in water at 50–60 1C and H2O2 solution, germination percentage and germination index of hard seeds were more than those of other seeds. 3.4. Seed ageing The germination percentage, germination index, and dehydrogenase activity of soft seeds decreased, and relative electric conductivity increased as ageing time increased; those of hard seeds did not have significant change after ageing for 1 day, but all changed significantly after 3 and more days (Table 1). There was a significantly negative relationship between germination index and days of ageing, but a positive relationship between the electrical conductivity rate and days of ageing for soft seeds (po0.05). The germination
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Table 1 Changes of GP, GI, REC and DA of Ammopiptanthus seeds after ageing for various days Species name
Ageing days
GP
GI
DA (OD490)
REC (%)
Hard seed
Control
Hard seed
Control
Hard seed
Control
Hard seed
Control
A. nanus
0 1 3 5 7
94.50 96.00 94.00 86.00 71.50
a a ab b c
85.00 78.50 71.50 62.50 48.00
a a b c d
24.23 25.17 24.69 23.15 20.43
a a a ab b
23.38 20.73 17.44 15.50 12.31
a a b bc c
42.33 44.67 51.67 60.33 66.33
a a b c c
47.33 57.67 67.33 78.00 84.00
a b c d d
0.46 0.42 0.38 0.36 0.30
a ab b b c
0.44 0.35 0.27 0.22 0.17
a b c cd d
A. mongolicus
0 1 3 5 7
90.50 88.50 87.00 77.50 70.50
a a ab bc c
85.00 84.50 73.00 64.00 45.50
a a b c d
23.87 23.25 22.32 20.78 20.13
a a a ab b
23.38 22.89 20.71 18.75 15.56
a a a ab b
48.00 51.33 62.00 65.00 71.33
a a b b c
51.00 64.00 77.67 79.00 86.33
a b c c
0.45 0.43 0.37 0.34 0.31
a a b bc c
0.46 0.37 0.32 0.24 0.18
a b bc d d
Note: GP, GI, REC, and DA means germination index, germination percentage, relative electrical conductivity, and dehydrogenase activity, respectively. The values with the same letter in the same column had no significant difference (po0.05, Fisher’s LSD test). The control was the soft seeds.
percentage, germination index, and dehydrogenase activity of hard seeds decreased and the electrical conductivity rate increased more slowly than those of soft seeds as ageing duration increased (Table 1). 4. Discussion and conclusions Our tests indicate that seed imbibition percentage of A. nanus and A. mongolicus decreased as seed moisture content and imbibition temperature decreased. The high hardseedness prevents immediate germination of Ammopiptanthus seeds in dry seasons since seeds do require a minimum soil moisture content to imbibe. We observed that imbibition percentage was lower in dark colored seeds of A. nanus and A. mongolicus. In other plant species, seed coat color darkens with increasing seed maturity (Argel and Humphryes, 1983), and seeds with unpigmented seed coat may deteriorate more rapidly and can be more susceptible to imbibition damage than those with pigmented seed coat (Abdullah et al., 1991; Asiedu and Powell, 1998). The present study also showed that as the seed coat darkened, the seed imbibition percentage decreased. Other researches have shown that soybean seeds with black seed coat had slower initial imbibition rates, higher resistance to field deterioration, and thicker and tougher seed coat than those with non-black seed coat (Chachalis and Smith, 2000; Krzyzanowski et al., 1999). Seed coats of plant species in 15 families, including Fabaceae, are temporarily impermeable to water and gases and do not germinate even if they are in conditions ideal for germination (Souza and Marcos-Filho, 2001). High hardseedness is the reason for seed coat-imposed dormancy (Bewley and Black, 1994), or physical dormancy (Baskin et al., 2000). Our ageing test using electrical conductivity of electrolytes leached from imbibed seeds and dehydrogenase activity of TTC solutions showed the hard seeds A. nanus and A. mongolicus can tolerate ageing and thus adverse storage conditions better than soft seeds. So the hard seeds had better storability than soft ones. There was hardseedness variation within seed population of A. nanus and A. mongolicus and the germination varied with hardseedness level (Fig. 2). Germination percentage and germination index of seeds rose gradually with increase in hardseedness level, but the hard seeds required pretreatments to relieve the constraint of seed coat before germination. The hardseedness variation with moisture content, seed coat color, and soaking duration could be necessary for the populations to persist in environments that change in both time and space, and could be a survival strategy to ensure that germination occurs under the most favorable environments. For germplasm conservation, the seeds with higher hardseedness should be preferred to those with lower hardseedness. In arid and semi-arid areas, many legume species have delayed hard-seed breakdown patterns with most seeds remaining hard until late autumn (Norman et al., 1998). Buried seeds of some legume species can survive
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30
100 80
ab
a
a
a
a
25
b
a
a
a
b
20 GI
60
GP(%)
ab c
40
15 10
20
5
0
0 CK1
CK2
CK3
CK4
CK5
H
CK1
CK2
CK3
CK4
CK5
H
Hardseedness level
Hardseedness level A. nanus 30
100
bc e
60
a
ab
25
d
20 f
GI
GP(%)
80
ab
cd
a
a
bc
d
15
40
10
20
5
0 CK1
CK2
CK3 CK4 Hardseedness level
CK5
H
CK1
CK2
CK3
CK4
CK5
H
Hardseedness level A. mongolicus
Fig. 2. Germination capability of seeds with various hardseedness levels. Values with the same letters on left corner are not significantly different at p ¼ 0.05 in Fisher’s LSD test.
as hard seeds for up to 12 years without significant loss of viability (Taylor and Ewing, 1988), which increases the seed longevity and population persistence. High hardseedness contributes to persistent soil seed bank by preventing germination from season rainfall and allows a certain proportion of a seed population to survive even in extended drought (Norman et al., 1998). It is also one of the most important adaptive and survival strategy of Ammopiptanthus species. The extended germination period caused by high hardseedness could improve the chances to get a good stand, since the hardseedness varies and this determines seed dormancy or readiness to germinate. Acknowledgments We thank Dr. Wang Zhangming and two anonymous referees for advice on data analysis and language revision. This research was funded by State Key Basic Research and Development Plan of China (G2000046803) and Key Natural Scientific Projects of Guangdong Province, China (021536). References Abdullah, W.D., Powell, A.A., Matthews, S., 1991. Association of differences in seed vigour in long bean (Vigna sesquipedalis) with testa colour and imbibition damage. Journal of Agricultural Science 116, 259–264. Argel, P.J., Humphryes, L.R., 1983. Environment effects on the seed development and the hardseededness in Stylosanthes hamata cv. Verano. I. Temperature. Australian Journal of Agricultural Research 34, 261–270. Asiedu, E.A., Powell, A.A., 1998. Comparisons of the storage potential of cultivars of cowpea (Vigna unguiculata) differing in seed coat pigmentation. Seed Science and Technology 26, 211–221. Baskin, J.M., Baskin, C.C., Li, X., 2000. Taxonomy, anatomy and evolution of physical dormancy. Plant Species Biology 15, 139–152. Bewley, J.D., Black, M., 1994. Seeds: Physiology of Development and Germination. Plenum Press, New York, pp. 147–197. Chachalis, D., Smith, M.L., 2000. Imbibition behavior of soybean (Glycine max (L.) Merril) accessions with different testa characteristics. Seed Science and Technology 28, 321–331. Cheng, S.H., 1959. Ammopiptanthus Cheng f. A new genus of Leguminosae from central Asia. Journal of Botany (USSR) 44, 1381–1386.
ARTICLE IN PRESS Q.-H. Yang et al. / Journal of Arid Environments 72 (2008) 263–269
269
Fu, L.G., 1989. The Rare and Endangered Plants in China. Shanghai Education Press, Shanghai. Krzyzanowski, F.C., Franc- aneto, J.B., Kaster, M., Mandarino, J.M.G., 1999. Metodologia para selec- a˜o de geno´tipos de soja com semente resistente ao dano mecaˆnico—relac- a˜o com o conteu´do de lignina. In: Resultados de pesquisa da Embrapa Soja, 1998. Londrina, EMBRAPA/CNPSo, pp. 213–214. Liu, G.H., 1998. Study on the endangered reasons of Ammopiptanthus mongolicus in the desert of Alashan. Bulletin Botanical Research 18, 341–345. Norman, H.C., Cocks, P.S., Smith, F.P., Nutt, B.J., 1998. Reproductive strategies in Mediterranean annual clovers: germination and hardseededness. Australian Journal of Agricultural Research 49, 973–982. Pan, B.R., Huang, S.P., 1993. Cytological study of the genus Ammopipthanthus. Acta Botanica Sinica 35, 314–317. Pan, B.R., Yu, Q.L., Yan, C., 1992. Study for the ecological environment and vulnerable reasons of the Ammopiptanthus nanus. Acta Phytecologia et Geobotanica Sinica 16, 276–282. Souza, F.H.D., Marcos-Filho, J., 2001. The seed coat as a modulator of seed–environment relationships in Fabaceae. Revista Brasileira de Botaˆnica 24, 365–375. Taylor, G.B., Ewing, M.A., 1988. Effect of depth of burial on the longevity of hard seeds of subterranean clover and annual medics. Australian Journal of Experimental Agriculture 28, 77–81. Yang, Q.H., Ge, X.J., Ye, W.H., Deng, X., Liao, F.L., 2004. Characteristics of Ammopiptanthus nanus seed and factors affecting its germination. Acta Phytoecologica Sinica 28, 651–656.