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Effects of environmental stress on seed germination and seedling growth of Salsola ferganica (Chenopodiaceae)
Acta Ecologica Sinica 36 (2016) 456–463
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Acta Ecologica Sinica journal homepage: www.elsevier.com/locate/chnaes
Effects of environmental stress on seed germination and seedling growth of Salsola ferganica (Chenopodiaceae) Yali Ma a,b,c, Jinghua Zhang b, Xiaorong Li b, Shiyue Zhang b, Haiyan Lan b,⁎ a b c
College of Resource and Environment, Xinjiang University, Urumqi 830046, China Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830046, China Xinjiang Education Institute, Urumqi 830043, China
a r t i c l e
i n f o
Article history: Received 3 November 2015 Received in revised form 18 January 2016 Accepted 16 May 2016 Keywords: Salsola ferganica Environmental stress Seed germination Seedling growth Desert annual halophyte
a b s t r a c t Plant growth and development are usually influenced by salt, drought, high or low temperature, strong illumination and other adverse factors, which may finally threaten the settlement and propagation of the species. Studies on seed germination behavior and seedling growth of annual halophyte plant living in harsh environment can help us to thoroughly understand the tolerance mechanism of desert plants. Salsola ferganica, an annual halophyte living in extreme desert habitat, has special morphological structure and characteristics in tolerance of stress. In the present study, we discussed the effects of different stress conditions, such as light, day/night temperature variation, salt (NaCl) and drought (PEG 6000), etc. on seed germination (SG) and seedling growth (SGr) of S. ferganica. Results showed that: (1) Light had a positive effect, while darkness had a negative effect on SG, which indicates that seed germination of S. ferganica depends on and is sensitive to light. (2) Monthly examination of SG from 2014 to 2015 showed that SG of seed without winged perianth (NWP) was apparently higher than seed with winged perianth (WWP) stored at room temperature (RT) or 4 °C. For WWP seeds, SG in light was significantly higher than that in darkness; SG also varied among seasons: in Spring, SG of seeds stored at RT was the lowest and so did it in summer with seeds stored at 4 °C. SG of seeds stored at both temperatures had no significant difference at the same month. Seed vigor agreed with SG behavior partly and there was no obvious difference between months. (3) The day/night temperature variation (D/NTV) applied significant effect on SG and SGr. SG decreased at lower D/NTV (e.g. 5 °C/15 °C) but increased at middle and higher D/NTV (e.g. 10 °C/20 °C, 15 °C/25 °C, 20 °C/30 °C) in light. In darkness, the SG decreased at higher D/NTV (e.g. 15 °C/ 25 °C, 20 °C/30 °C) but increased at lower and middle D/NTV (e.g. 5 °C/15 °C, 10 °C/20 °C). 10 °C/20 °C D/NTV had positive effect on SG in both light and darkness; germination rate was promoted at higher D/NTV in both light and darkness. SGr was promoted at lower D/NTV while inhibited at higher D/NTV. (4) SG at lower concentration of NaCl (b 100 mmol L−1, osmotic pressure (OP) ≥ −500 kPa) and PEG (b150 g L−1, OP ≥ −300 kPa) was similar to the control (OP 0 kPa); however SG at higher concentration of NaCl (≥500 mmol L−1, OP b −2478 kPa) and PEG (≥200 g L−1, OP b −300 kPa) significantly decreased but still exceeded 35%, indicating that S. ferganica is salt- and drought-tolerant. When considered of the SG between NaCl and PEG treatment at the same level of OP, which was much better with NaCl than that with PEG. Taken together, we speculate that S. ferganica should employ ‘cautious strategy’ in germination, which means that under favorable conditions with light, temperature, water, etc., S. ferganica can germinate actively and get enough seedlings developing into adult plants; whereas under unfavorable conditions, the seeds may not germinate or germinate in a small amount to replenish soil seed bank. © 2016 Ecological Society of China. Published by Elsevier B.V. All rights reserved.
1. Introduction Plant growth and development are usually influenced by salt, drought, high or low temperature, strong illumination and other adverse factors, which may finally threaten the settlement and thriving of the species. To cope with this, desert plants have evolved a variety ⁎ Corresponding author. E-mail address: [email protected] (H. Lan).
http://dx.doi.org/10.1016/j.chnaes.2016.09.008 1872-2032/© 2016 Ecological Society of China. Published by Elsevier B.V. All rights reserved.
of adaptation strategies, especially for the annual desert plant species [1]. The adaptability of seed germination and seedling establishment to the heterogeneous environment is pivotal for succession of the population and propagation of species. Various environmental factors, e.g. light, temperature, salt, drought, etc. have great effects on seed germination and seedling growth of annual desert plant. Seed germination and seed vigor can be significantly influenced by changes of afterripening time and temperature [1,2]. Seed vigor is formed in the dehydration process of seed development [3], because mRNA, storage
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proteins, the late embryonic development abundance proteins (LEAs) and heat shock proteins will be changed upon dehydration, which results in variation of seed vigor [4,5]. Temperature can largely change the biological metabolism and enzymatic activity of seed and consequently influence the germination behavior [6,7]. In desert habitat, large temperature fluctuations exist, and which result in broad temperature variation between day and night. Corresponding to this, seed germination of the desert plant is of wide temperature range, but different species show the diverse optimum germination temperature [2,8]. Previous research showed that Nitraria tangutorum in the family of Zygophyllaceae could germinate in the range of − 15 °C– 30 °C, and the best germination was at a day/night temperature variation (D/NTV) of 25 °C/35 °C [9]. Suaeda aralocaspica (Chenopodiaceae) can germinate in a range of 5 °C–35 °C, and the optimum D/NTV is 15 °C/25 °C [10,11]; whereas Salsola korshinskyi (Chenopodiaceae) which grows in the similar habitat with S. aralocaspica shows the greatest germination percentage of D/NTV at 0 °C/10 °C [12]. Light is also an important factor for seed germination of plant species which are sensitive to light [8,13]. For some plants, light has crucial effect and can significantly promote seed germination [14,15]. On the contrary, some species will have the best germination in darkness [16–18]. Still some other species are insensitive to light during germination [10,19–21]. In addition to light, water is also crucial for seed germination. There is a limited and large variation of precipitation, which results in great changes of the soil humidity between different seasons or years in desert, so the moisture is becoming a necessary factor for seed germination [22,23]. Effective water-imbibition of seeds launches seed germination, which is in turn affected by the adjustment of the osmotic pressure generated inside soil and plant [3,24,25]. Seed physiological & biochemical aspects and the osmotic pressure are changed by salt and drought stress, which consequently affect seed germination [2,8,26]. Salt stress can change the integration and permeability restoration of cell membrane, induce the denaturation of proteins in the cell, inhibit DNA synthesis, which has dual function of osmotic stress and ion poisoning [27–30]. Drought stress is usually simulated by polyethylene glycol 6000 (PEG 6000) [31,32]. The previous study showed that effects of isotonic PEG and NaCl on plant was significantly different, which may be caused by the ion regulation mechanism [28,33]. Desert plant seeds often have bracts and winged perianths, e.g. seeds of species in genus of Atriplex, Salsola, Haloxylon [2,12,21,34,35], etc. On the one hand, winged perianth is beneficial for seed dispersal in the desert; On the other hand, it applies certain negative effect in seed germination, which may change with different abiotic factors [34,36,37]. Both of these aspects can effectively adjust the balance between seeds germination at the best time and seeds persistence short or long-term in soil seed bank in early spring [13]. Not only germination but seed vigor and seedling growth can all reflect the effectiveness of germination behavior and the result of stress [2,25]. Abiotic factors, e.g. temperature, salt, drought, etc., impact on plant height, root length of seedlings [10,13]. Previous research showed that a high consistency existed between plant height of early seedling and seed tolerance, rather than root length variation [16,20]. Alive mature seeds will be forced to become dormant or induced into secondary dormancy because of the seed (fruit) structure, e.g. winged perianth, etc. and abiotic stress, e.g. salt and drought, which is one of the adaptive strategy of desert plants in germination [1,2,38, 39]. However, different desert plants evolve different ways to adapt environment [6,22,40], so systematically studying the effect of abiotic factors, e.g. light, temperature, salt, drought, etc., on seed germination and seedling growth should contribute to further understanding of the unique mechanism of the desert plant to adapt to the heterogeneous circumstance in the early stage. Salsola ferganica, an annual desert pioneer halophyte of Salsola genus in Chenopodiaceae family, referred as “natural desalter” [29,41]. Through long-term observation, we found that S. ferganica seeds which have membranous winged perianth could germinate under dense salt crust in early spring when snow
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was melting. It suggests that seed and seedling of S. ferganica are of strong stress tolerance, but the systematic and thorough study of the stress effects on the seed germination and seedling growth is limited. Based on the above situation, in the present study, we investigated the effects of different stress conditions, such as light, storage temperature, day/night temperature variation, salt (NaCl) and drought (PEG 6000), etc. on seed germination (SG) and seedling growth (SGr) of S. ferganica, which may facilitate to further explore the mechanism and adaption strategy of desert plants in early stage, and provide scientific evidence for ecological restoration and reconstruction of the natural environment. 2. Materials and methods 2.1. Seed collection Salsola ferganica usually germinates in late March and early April, blooms in middle of July and August, bears seeds in late September and early October in the natural habitat in Junggar Basin, Xinjiang, China. Seeds of S. ferganica in the present study were collected in October 2013 at Wujiaqu 103 regiment (44°29′821″ N, 87°31′181″ E; 429 m H), Xinjiang. Harvested seeds were air-dried for 2 weeks under room conditions: 18–26 °C, 8–20% relative humidity, then cleaned to remove the impurities and stored in a dry cardboard case in room temperature; meantime, part of seeds were stored at 4 °C. In the present study, seed with membranous appendage (like wing) is named as seed with winged perianth (WWP); seed removed membranous appendages is named as seed without winged perianth (NWP). 2.2. Seed germination experiments Mature intact seeds (WWP, the radius is 3.0–4.5 mm) stored at room temperature or 4 °C were used in seed germination experiments. Four replicates with 30 seeds of each were placed on two layers of filter paper in 9-cm Petri dishes, in which 7 mL of distilled water were added. All Petri dishes were sealed with cling film, and placed in an illuminated incubator (RXZ-500D-LED; Jiangnan Apparatus Manufactory, China), with a 12 h light/12 h dark photoperiod (light flux: approx.100 μmol m−2 s−1), 25 °C, 50% relative humidity. For incubation in darkness, seeds were prepared in the dark room and packaged with foil paper, and checked for germination only at the end of the experiment to avoid exposure to any light during germination. It was considered to be germinated as the radicle of a seed was at least half length of seed without winged perianth. Seed germination was recorded every 24 h for 15 d. The height and root length of seedlings were examined on the 15th day and calculated by Image J 1.47 V (Wayne Rasband National Institutes of Health, USA) with 10 plants in each treatment. Seed germination percentage (G) was calculated as: G = Gi/ Gt × 100%, where Gi represents the number of germinated seeds 15 d later; Gt represents the total number of seeds of each replicate. Seed germination rate (T50/d) is the time (day) that germination percentage reaches the half of the final germination percentage in light [42,43]. 2.3. Test for seed vigor with 2,3,5-triphenyltetrazolium chloride (TTC) After soaking in sterilized distilled water at 28 °C for 20 h, S. ferganica seeds were stained with 0.1% TTC [0.1 g TTC dissolved in 100 mL of 0.1 mol L−1 phosphate buffer (PBS, pH 7.0)] for 12 h at 30 °C in an incubator (EYELA NDO-400, Tokyo Rikakikai C., LTD in Shanghai) in darkness. After being rinsed with distilled water for three times, the treated seeds were examined under stereomicroscope to check the embryo vigor. Seed vigor percentage was calculated according to Zhang and Zhai (2003) [44]. Seeds with stronger vigor were stained in dark red, weaker vigor in pink, inactive seeds would not be stained. Four replicates with 30 seeds of each were applied for one treatment.
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2.4. Seed germination experiments 2.4.1. Light treatment To determine the effect of light on seed germination, white light (wavelength 380–750 nm) and darkness were used in experiment. For seeds in darkness, the Petri dishes were wrapped with foil paper compared to those in light only sealed with cling film [45]. 2.4.2. Day/night temperature variation (D/NTV) treatment Variations of day/night temperature regimes [5 °C in night/15 °C in day (mimic early spring daily temperature regime in natural habitat), 10 °C/20 °C (spring with lower temperature), 15 °C/25 °C (spring with higher temperature), 20 °C/30 °C(late spring)] were applied in seed germination in an illuminated incubator (RXZ-500D-LED; Jiangnan Apparatus Manufactory, China), and subjected to a 12 h in dark (lower temperature)/12 h in light (higher temperature) photoperiod. 2.4.3. Salt and drought treatment Different concentrations of NaCl (0, 50, 100, 300, 500, 700 and 1000 mmol L− 1, corresponding to osmotic pressure − 248, − 496, − 1487, − 2478, − 3469, − 4955 kPa respectively) were applied in seed germination [46]. For PEG treatment, different concentrations of PEG 6000 solution (0, 25, 50, 100, 150, 200 and 300 g L−1, corresponding to osmotic pressure −20, −50, −150, −300, −500, −1000 kPa) were used in seed germination [31]. Sterilized distilled water (osmotic pressure 0 kPa) treatment was used as control. 2.5. Statistical analysis All data were expressed as mean ± standard deviation. One- or twoway ANOVA was used to analyze variable temperature, NaCl and PEG treatment on seed germination and seedling growth, as well as variable temperature on germination rate with the GraphPad Prism Version 5.01 for Windows (GraphPad Software, SanDiego, CA, USA). If significant main effects existed, differences were tested by a multiple comparison Tukey test at 0.05, 0.01, 0.001 significance level. Three-way ANOVA was used to analyze seed type, light, storage time and their interaction
on seed germination of S. ferganica with the SPSS Version 17.0 for Windows (SPSS Inc., Chicago, IL, USA). 3. Results 3.1. Effect of storage time (month) on seed germination and seed vigor 3.1.1. Seed germination The germination trend of S. ferganica seeds at room temperature (RT) and 4 °C storage showed that regardless in light or darkness, seeds without winged parianth (NWP) had high germination percentage with the time increasing; whereas the germination of seeds with winged parianth (WWP) significantly decreased in darkness, which suggests seeds of S. ferganica need light in germination. Besides, germination percentage of NWP seeds was significantly higher than that of WWP seeds at the same storage condition, especially in darkness (Fig. 1). At RT, the germination of NWP seeds and WWP seeds gradually increased and reached the highest percentage in October and kept stable then (Fig. 1A,B); at 4 °C, before November, the germination of S. ferganica seeds was high, while decreased gradually after (Fig. 1C,D). Under light, NWP rather than WWP seeds kept stably higher germination percentage at RT after October (Fig. 1A). NWP seeds showed decreased germination after October; whereas WWP seeds kept higher germination percentage before Feburary of next year and decreased gradually after (Fig. 1C). In darkness, NWP seeds showed similar germination trend under the same storage temperature; whereas WWP seeds had similar germination trend under different storage temperature (Fig. 1B,D). Our results suggest that light applies significant effect to S. ferganica seeds; low temperature delays germination to some extent. Three-way ANOVA analysis (Table 1) of seed type, light, storage time and their interaction on seed germination at different temperatures of S. ferganica showed that regardless at RT or 4 °C, seed types or light types had significant interaction (P b 0.001), for storage time (month), only 4 °C showed significant interaction (P = 0.095 for RT; P b 0.05 for 4 °C); significant interaction existed between seed types and light types both at RT and 4 °C (P b 0.001), while between seed types and
Fig. 1. Change of germination percentage of S. ferganica seeds stored at different temperatures from 2014 to 2015. (A, B) Room tempr. & in light/dark: seeds were stored under room temperature and germinated in light/dark; (C, D) 4 °C & in light/dark: seeds were stored under 4 °C and germinated in light/dark. tempr.: abbreviation of temperature.
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Table 1 Three-way ANOVA analysis of seed type, light, storage time and their interaction on seed germination at different temperatures of S. ferganica. Source of variation
Seed type (S) Light (L) Storage time (Month, M) S ×L S ×M L ×M S ×L ×M
Room temperature
4 °C
d.f.
SS
MS
F-value
P-value
d.f.
SS
MS
F-value
P-value
1 1 12 1 12 12 12
19.352 13.613 0.652 10.199 0.844 0.478 0.676
19.352 13.613 0.054 10.199 0.070 0.040 0.056
571.401 401.950 1.605 301.145 2.076 1.177 1.663
b0.001 b0.001 0.095 b0.001 0.021 0.304 0.080
1 1 12 1 12 12 12
11.855 12.404 1.341 7.714 0.980 0.853 0.957
11.855 12.404 0.112 7.714 0.082 0.071 0.080
212.743 222.600 2.005 138.427 1.466 1.276 1.431
b0.001 b0.001 0.027 b0.001 0.143 0.238 0.157
storage time (month), significant interaction only existed at RT (P = 0.021 for RT; P = 0.143 for 4 °C), no significant interaction was observed between light types and storage time (month) (P = 0.304 for RT; P = 0.238 for 4 °C); no significant interaction existed among seed types, light types and storage time (month) at RT or 4 °C (P = 0.08 for RT; P = 0.157 for 4 °C). Analysis of germination rate of WWP and NWP seeds under varying storage temperatures showed that germination rate of WWP seeds was first increased and then gradually decreased, however, fluctuation existed between different months (Fig. 2). Germination rate of NWP seeds kept stably higher trend. 3.1.2. Seed vigor TTC (2,3,5-triphenyl-2H-tetrazolium chloride) staining can exhibit the status of seed vigor, vigorous seeds are stained with dark red color, lower vigor seeds are stained with pink color, inactive seeds cannot be stained (Fig. 3). S. ferganica seeds were stained with TTC from Oct. 2013, which showed a lower vigor of the newly-mature seeds, and then gradually increased and reached the highest in Apr.–May (spring) of the next year, which showed the completion of after-ripening of seeds (Fig. 4). With the temperature rising, seed vigor presented decrease trend, and had a significant reduction in Jun. of the next year, which may be related to the summer dormancy under high temperature. From Nov. 2014 to Mar. 2015, seed vigor gradually decreased. A correlationship existed between seed vigor monthly change (Fig. 4) to seed germination (Fig. 1). 3.2. Effect of day/night temperature variation (D/NTV) on seed germination and seedling growth 3.2.1. Seed germination D/NTV had little effect on NWP seed germination of S. ferganica, while applied significant effect to that of WWP seeds (Fig. 5). In light (Fig. 5A), NWP seeds at 5 °C day/15 °C night, 10 °C/20 °C, 15 °C/25 °C, 20 °C/30 °C showed high germination percentage, which was more than 95%; while germination of WWP seeds was promoted under
Fig. 2. Change of germination rate between months of S. ferganica seeds stored at different temperatures from 2014 to 2015. Room tempr. & NWP/WWP: seeds without/with winged perianth were stored at room temperature; 4 °C & NWP/WWP: seeds without/with winged perianth were stored at 4 °C. tempr.: abbreviation of temperature.
various D/NTV (≥ 85% compared to the control 80%) except for 5 °C/15 °C (about 45%). In darkness (Fig. 5B), there had little effect of D/NTV on NWP seed germination (≥ 95%) except for 20 °C/30 °C (about 70%); WWP seed germination was significantly promoted at 5 °C/15 °C and 10 °C/20 °C D/NTV (about 67% and 80%) compared to control (17%), while no significant effect was at 15 °C/25 °C and 20 °C/30 °C. As shown in Fig. 5, although lower D/NTV significantly affected seed germination of S. ferganica in light, it could partially counteract the inhibition effect of darkness on seed germination. Analysis of germination rate under D/NTV showed that WWP seed germination rate increased with the rising of D/NTV (Fig. 6), germination at 5 °C/15 °C was the slowest, 20 °C/30 °C was the fastest, D/NTV had little effect on germination rate of NWP seeds. 3.2.2. Seedling growth Analysis of seedling growth of S. ferganica under D/NTV showed that, regardless in light or darkness, lower D/NTV (5 °C/15 °C) promoted NWP and WWP seedling growth (8.686 mm and 8.768 mm for height; 6.233 mm and 5.744 mm for root length) (Fig. 7A,C); NWP seedling growth reduced with the rising of D/NTV. In light, NWP seedling growth was better than that of WWP, especially at 15 °C/25 °C. In darkness, WWP seedling growth was the best at 5 °C/15 °C, while NWP seedling growth was promoted at various D/NTV (especially at 10 °C/20 °C) except for 20 °C/30 °C (Fig. 7B,D). NWP seedling growth was better in darkness than that in light. 3.3. Effect of salinity or drought on seed germination and seedling growth Under lower NaCl concentration [100 mmol L−1, −496 kPa osmotic pressure (OP)], WWP seed germination had no significant difference compared to control (Fig. 8A); under middle NaCl concentration (300 mmol L−1, − 1487 kPa OP), seed germination reduced to about 50%; under higher NaCl concentration (500 mmol L−1, − 2478 kPa OP), seed germination remained about 40%, which suggests S. ferganica can tolerate higher salinity. Under lower concentration of PEG (b150 g L− 1, ≥− 300 kPa OP), WWP seed germination had no significant difference compared to control (Fig. 8B); under middle concentration (200 g L−1, −500 kPa OP), seed germination remained more than 60%; under higher concentration (300 g L−1, −1000 kPa OP), seed germination decreased significantly. Although 100 mmol L−1 NaCl and 200 g L−1 PEG had nearly the same
Fig. 3. TTC staining of S. ferganica seeds. (A) Dark red seeds; (B) pink seeds; (C) unstained seeds.
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Fig. 4. The vigor of S. ferganica seeds stored at room temperature by TTC method from 2014 to 2015. Rs: dark red seeds (stronger vigor); Ps: pink seeds (weaker vigor); Us: unstained seeds (inactive vigor).
OP, seed germination and seedling growth of the former were significantly greater than that of the latter. Analysis of seedling growth of S. ferganica showed that effect of NaCl or PEG treatment was greater on NWP than that of WWP seedling growth, especially PEG (Fig. 9). With the rising of NaCl and PEG concentration, both NWP and WWP seedling height and root length were inhibited (Fig. 9A,B,C,D). Under similar OP of NaCl or PEG treatment, PEG had greater effect on NWP seedling growth while had little effect on that of NWP than that of NaCl. 4. Discussion The adaptability of seed germination and seedling growth is pivotal for plant establishment, succession of the population and propagation of species to the heterogeneous environment. In the present study, we investigated the effects of different stresses on seed germination (SG) and seedling growth (SGr) of S. ferganica, an annual halophyte in desert, which may facilitate to explore the mechanism and adaptation strategy in early stage of plant growth. Our results showed that light quality, day/ night temperature variation, and winged perianth etc., applied significant effects on SG and SGr; S. ferganica SG and SGr could tolerate high salinity and drought. 4.1. Seed germination of S. ferganica was sensitive to light Desert halophytes show different sensitivity to light in seed germination. For some plant species, light has crucial function on SG and SGr [7,46,47], e.g. Cyperus arenarius [8] and Cyperus conglomeratus [18]
Fig. 6. Effect of day/night temperature variation (D/NTV) on germination rate of S. ferganica in light. Lowercase letters are variance analysis results of germination rate, different letters show significant difference (P b 0.05). *, **, *** indicate significant difference by t-test (P b 0.05, 0.01, 0.001). Horizontal ordinate represents D/NTV: 25 °C, germination at 25 °C for 24 h; 5 °C/15 °C, germination at 5 °C for 12 h in darkness and at 15 °C for 12 h in light. The sets of other D/NTV were similar with 5 °C/15 °C.
in Cyperaceae family, Sporobolus ioclados [48] (Gramineae), Tamarix aucheriana [49] (Tamaricaceae), Halostachys caspica [15] (Chenopodiaceae), etc., which can achieve much better germination in light compared to that in darkness. In our present study, germination of S. ferganica seeds with winged perianth was sensitive to light, seed germination was much higher in light than in darkness under the same treatment. On the contrary, the germination of some desert species, e.g. Panicum turgidum [17] (Gramineae), Tanacetum cinerariifolium [16] (Compositae), etc., get the highest germination percentage in darkness [16–18], which suggests the characteristics of light independence [47]. Still some other species, e.g. Suaeda salsa [20,50], Suaeda aralocaspica [10,11] and Salsola affinis [21] in Chenopodiaceae etc., present similar seed germination behavior in light or darkness, which is insensitive to light in germination. Light applies effect on both seed germination and early seedling growth, however, they might have completely different responses in light requirement and light sensitivity [47]. Requirement of different light wavelength in seed germination and seedling photomorphogenesis is probably related to different adaptation strategies of various desert plant species [13,43]. In our study, the seedling of S. ferganica grew better in darkness than that in light, which suggests that darkness may promote hypocotyl elongation and radicle protrusion at early stage of germination. The effects of light with different wavelength on SG and SGr depend on plant species and genetic factors [47,51], e.g. seed germination of Lactuca sativa is red light-dependent [52]. The previous research showed that seed germination affected by different light qualities may be related to the phytochromes [53,54].
Fig. 5. Effect of variable temperature on germination of S. ferganica in light (A) and dark (B). Lowercase letters are variance analysis results of seed germination, different letters show significant difference (P b 0.05). Horizontal ordinate, daily temperature regime: 25 °C, seed germination temperature kept at 25 °C for 24 h; 5/15 °C, seed germination temperature kept at 5 °C in dark for 12 h, and kept at 15 °C in light. The sets of other daily temperature regimes were similar with 5/15 °C.
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Fig. 7. Effect of D/NTV on seedling growth of S. ferganica in light (A,B) and darkness (C,D). Capital and lowercase letters are variance analysis results of seedling height and root length, different letters show significant difference (P b 0.05). *, **, *** indicate significant difference by t-test (P b 0.05, 0.01, 0.001). Horizontal ordinate represents D/NTV: 25 °C, germination at 25 °C for 24 h; 5 °C/15 °C, germination at 5 °C for 12 h in darkness and at 15 °C for 12 h in light. The sets of other D/NTV were similar with 5 °C/15 °C.
4.2. Appropriate temperature and its variation were important for seed germination and seedling growth of S. ferganica Temperature as one of the important factor of environmental stress plays vital role for seed germination and early seedling growth [13,38]. Temperature fluctuates largely among different years, and shows drastic change between day and night, which is especially true in early spring as snow melting in desert habitat [6,23]. The desert plant can respond to temperature variation and germinate rapidly [2,22,38]. Some desert plant can germinate in a wide temperature range, Hordeum jubatum [55] (Gramineae) and Haloxylon stocksii [56] (Chenopodiaceae) have the highest germination percentage at a day/night temperature variation of 5 °C/20 °C and 10 °C/30 °C respectively. In the present study, S. ferganica showed the optimum germination at 10 °C/20 °C. The desert plants such as Suaeda physophora [57], Atriplex stocksii [58], Salsola affinis [8,21] all have the highest germination percentage at a day/night temperature variation of 10 °C/20 °C. Similar range of the optimum germination temperature of plants which lived in similar habitat is a characteristic of convergence, in which plant germinates in mid April when temperature is rising (corresponding to the optimum germination range), suggesting the adaptability of these plant species to early spring weather [2,7,13,38].
Seed germination can significantly be affected by changes of environmental temperature in seed storage. In the present study, S. ferganica seeds showed reduced germination percentage in June–August, which may be in connection with summer dormancy of this species under high temperature [59]. Poor germination of Limonium stocksii [60] (Plumbaginaceae) and Atriplex centralasiatica [61] (Chenopodiaceae) was observed at high day/night temperature range of 25 °C/35 °C. Seed vigor can be significantly influenced by storage time, storage temperature and internal components of seed itself [7,62,63]. Seed vigor of Haloxylon (Chenopodiaceae) dramatically decreased after 9–12 months storage at room temperature, and poor germination was present after 17 months [15]. In our experiment, S. ferganica seed remained high seed vigor (germination more than 90%) after 13–15 months storage, which suggests that S. ferganica seeds can keep vigor for longer. 4.3. Seed accessory structure applied special ecological role in seed germination of S. ferganica Desert plant species usually develop accessory structures with seeds to acclimate environment. The winged perianth of seed has an important role in ecological adaptability [7,36]. It can contribute to seed dispersal, and force seeds into secondary dormancy [37,59]. However, as
Fig. 8. Effect of NaCl or PEG on germination of S. ferganica. Lowercase letters are variance analysis results of NaCl or PEG on germination, different letters show significant difference (P b 0.05). *, **, *** indicate significant difference by t-test (P b 0.05, 0.01, 0.001).
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Fig. 9. Effect of NaCl (A,C) or PEG (B,D) on seedling growth of S. ferganica in light (A,B) and darkness (C,D). Capital and lowercase letters are variance analysis results of seedling height and root length, different letters show significant difference (P b 0.05). *, **, *** indicate significant difference by t-test (P b 0.05, 0.01, 0.001).
the natural mechanical barrier between seed and its original surrounding, winged perianth can assist desert seeds to choose optimum chance to germination, which fits for “bet-hedging or conservative strategy” of seed germination [2,7,8,13,38]. In the present study, S. ferganica germination percentage of NWP seeds was higher than WWP seed under the same condition. A number of desert plant seeds with winged perianth or bract are of such characteristics [12,21,36,64]. Probably NWP seeds can quickly respond to light without mechanical block of winged perianth and germinate rapidly. In addition, endogenous hormones or chemicals in winged perianth may apply effect on seed germination [2,59]. 4.4. Seed germination and seedling growth of S. ferganica were tolerant to salt and drought stress Whether desert halophyte can survive in saline environment or not depends on seed and seedling responses to salt and drought stress [2,26]. Salt tolerance is much different among halophytes. Seed germination of Suaeda aralocaspica was more than 10% at 1400 mmol L−1 NaCl [10,11]; whereas that of Lepidium latifolium (Cruciferae) [65] and Limonium iconicum [66] (Plumbaginaceae) is almost 0 at 300 mmol L−1 NaCl. In our study, S. ferganica could germinate more than 3% at 700 mmol L−1 NaCl, which suggests that S. ferganica is relatively salt-tolerant. For desert halophytes, lower NaCl concentration can promote seed germination, however, higher NaCl concentration may force them into secondary seed dormancy [7,13,59]. Halophytes in Haloxylon genus [15,56], Suaeda genus [10,11,20,50], Salsola genus [12,21] (including S. ferganica in the present study) present similar salt tolerance in seed germination. Besides, our results showed that S. ferganica was drought-tolerant, which had similar trend with salt tolerance, while seed germination and seedling growth under NaCl treatment were significantly better than that of PEG treatment in which they shared the same osmotic pressure. It suggests that salt ions may relieve the effect of osmotic stress [27,28]. Our results agreed with the previous report which showed that the stress effect of PEG was significantly greater than that of NaCl [33]. 5. Conclusion Abiotic factors e.g. salt, drought, day/night temperature variation, and intense light irradiation etc., seriously influence desert plant
growth, especially in the critical periods of seed germination and seedling growth. Germination behavior and seed dormancy strategy have great effect on whole life of plants, which in turn impact population establishment and species propagation [2,7,8,13,38,59]. Our results showed that S. ferganica needed light in seed germination; the winged perianth applied certain negative effect in germination, while it also could adjust the balance between seed germination and dormancy in the proper time; appropriate day/night temperature variation promoted S. ferganica seed germination, while low temperature delayed germination process in some extent; seed germination and seed vigor of S. ferganica gradually decreased with the storage time increasing; seeds and seedlings of S. ferganica were salt- and drought-tolerant. Based on the above result, we speculate that S. ferganica employs ‘cautious strategy’ in germination, which means that under favorable conditions of light, temperature, water, etc., S. ferganica can germinate actively and get enough seedlings developing into adult plants; whereas under unfavorable conditions, seeds may not germinate or germinate in a small amount to replenish soil seed bank [2,7]. Acknowledgements This work was supported by the Project for Scientific Research Project in higher education of Xinjiang Uygur Autonomous Region (XJEDU2014S084); National Natural Science Foundation of China (31060027; 31260037; 31460043); Project for Training Young Talents of Xinjiang Uygur Autonomous Region (2013721013). References [1] Y. Zhang, L.G. Xue, T.P. Gao, L. Jin, L.Z. An, Research advance on seed germination of desert plants, J. Desert Res. 25 (1) (2005) 106–112. [2] X.X. Qu, Z.Y. Huang, The adaptive strategies of halophyte seed germination, Acta Ecol. Sin. 25 (9) (2005) 2389–2398. [3] L. Rajjou, M. Duval, K. Gallardo, J. Catusse, J. Bally, C. Job, D. Job, Seed germination and vigor, Annu. Rev. Plant Biol. 63 (3) (2012) 507–533. [4] Q. Sun, J.H. Wang, B.Q. Sun, Advances on seed vigor physiological and genetic mechanisms, Sci. Agric. Sin. 40 (1) (2007) 48–53. [5] Z.H. Li, J.H. Wang, Advances in research of physiological and molecular mechanism in seed vigor and germination, Sci. Agric. Sin. 48 (4) (2015) 646–660. [6] C.C. Baskin, J.M. Baskin, Germination ecophysiology of herbaceous plant species in a temperate region, Am. J. Bot. 75 (2) (1988) 286–305. [7] C.C. Baskin, J.M. Baskin, Seeds: Ecology, Biogeography and Evolution of Dormancy and Germination, Academic Press, San Diego, 1998.
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Acta Ecologica Sinica journal homepage: www.elsevier.com/locate/chnaes
Effects of environmental stress on seed germination and seedling growth of Salsola ferganica (Chenopodiaceae) Yali Ma a,b,c, Jinghua Zhang b, Xiaorong Li b, Shiyue Zhang b, Haiyan Lan b,⁎ a b c
College of Resource and Environment, Xinjiang University, Urumqi 830046, China Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830046, China Xinjiang Education Institute, Urumqi 830043, China
a r t i c l e
i n f o
Article history: Received 3 November 2015 Received in revised form 18 January 2016 Accepted 16 May 2016 Keywords: Salsola ferganica Environmental stress Seed germination Seedling growth Desert annual halophyte
a b s t r a c t Plant growth and development are usually influenced by salt, drought, high or low temperature, strong illumination and other adverse factors, which may finally threaten the settlement and propagation of the species. Studies on seed germination behavior and seedling growth of annual halophyte plant living in harsh environment can help us to thoroughly understand the tolerance mechanism of desert plants. Salsola ferganica, an annual halophyte living in extreme desert habitat, has special morphological structure and characteristics in tolerance of stress. In the present study, we discussed the effects of different stress conditions, such as light, day/night temperature variation, salt (NaCl) and drought (PEG 6000), etc. on seed germination (SG) and seedling growth (SGr) of S. ferganica. Results showed that: (1) Light had a positive effect, while darkness had a negative effect on SG, which indicates that seed germination of S. ferganica depends on and is sensitive to light. (2) Monthly examination of SG from 2014 to 2015 showed that SG of seed without winged perianth (NWP) was apparently higher than seed with winged perianth (WWP) stored at room temperature (RT) or 4 °C. For WWP seeds, SG in light was significantly higher than that in darkness; SG also varied among seasons: in Spring, SG of seeds stored at RT was the lowest and so did it in summer with seeds stored at 4 °C. SG of seeds stored at both temperatures had no significant difference at the same month. Seed vigor agreed with SG behavior partly and there was no obvious difference between months. (3) The day/night temperature variation (D/NTV) applied significant effect on SG and SGr. SG decreased at lower D/NTV (e.g. 5 °C/15 °C) but increased at middle and higher D/NTV (e.g. 10 °C/20 °C, 15 °C/25 °C, 20 °C/30 °C) in light. In darkness, the SG decreased at higher D/NTV (e.g. 15 °C/ 25 °C, 20 °C/30 °C) but increased at lower and middle D/NTV (e.g. 5 °C/15 °C, 10 °C/20 °C). 10 °C/20 °C D/NTV had positive effect on SG in both light and darkness; germination rate was promoted at higher D/NTV in both light and darkness. SGr was promoted at lower D/NTV while inhibited at higher D/NTV. (4) SG at lower concentration of NaCl (b 100 mmol L−1, osmotic pressure (OP) ≥ −500 kPa) and PEG (b150 g L−1, OP ≥ −300 kPa) was similar to the control (OP 0 kPa); however SG at higher concentration of NaCl (≥500 mmol L−1, OP b −2478 kPa) and PEG (≥200 g L−1, OP b −300 kPa) significantly decreased but still exceeded 35%, indicating that S. ferganica is salt- and drought-tolerant. When considered of the SG between NaCl and PEG treatment at the same level of OP, which was much better with NaCl than that with PEG. Taken together, we speculate that S. ferganica should employ ‘cautious strategy’ in germination, which means that under favorable conditions with light, temperature, water, etc., S. ferganica can germinate actively and get enough seedlings developing into adult plants; whereas under unfavorable conditions, the seeds may not germinate or germinate in a small amount to replenish soil seed bank. © 2016 Ecological Society of China. Published by Elsevier B.V. All rights reserved.
1. Introduction Plant growth and development are usually influenced by salt, drought, high or low temperature, strong illumination and other adverse factors, which may finally threaten the settlement and thriving of the species. To cope with this, desert plants have evolved a variety ⁎ Corresponding author. E-mail address: [email protected] (H. Lan).
http://dx.doi.org/10.1016/j.chnaes.2016.09.008 1872-2032/© 2016 Ecological Society of China. Published by Elsevier B.V. All rights reserved.
of adaptation strategies, especially for the annual desert plant species [1]. The adaptability of seed germination and seedling establishment to the heterogeneous environment is pivotal for succession of the population and propagation of species. Various environmental factors, e.g. light, temperature, salt, drought, etc. have great effects on seed germination and seedling growth of annual desert plant. Seed germination and seed vigor can be significantly influenced by changes of afterripening time and temperature [1,2]. Seed vigor is formed in the dehydration process of seed development [3], because mRNA, storage
Y. Ma et al. / Acta Ecologica Sinica 36 (2016) 456–463
proteins, the late embryonic development abundance proteins (LEAs) and heat shock proteins will be changed upon dehydration, which results in variation of seed vigor [4,5]. Temperature can largely change the biological metabolism and enzymatic activity of seed and consequently influence the germination behavior [6,7]. In desert habitat, large temperature fluctuations exist, and which result in broad temperature variation between day and night. Corresponding to this, seed germination of the desert plant is of wide temperature range, but different species show the diverse optimum germination temperature [2,8]. Previous research showed that Nitraria tangutorum in the family of Zygophyllaceae could germinate in the range of − 15 °C– 30 °C, and the best germination was at a day/night temperature variation (D/NTV) of 25 °C/35 °C [9]. Suaeda aralocaspica (Chenopodiaceae) can germinate in a range of 5 °C–35 °C, and the optimum D/NTV is 15 °C/25 °C [10,11]; whereas Salsola korshinskyi (Chenopodiaceae) which grows in the similar habitat with S. aralocaspica shows the greatest germination percentage of D/NTV at 0 °C/10 °C [12]. Light is also an important factor for seed germination of plant species which are sensitive to light [8,13]. For some plants, light has crucial effect and can significantly promote seed germination [14,15]. On the contrary, some species will have the best germination in darkness [16–18]. Still some other species are insensitive to light during germination [10,19–21]. In addition to light, water is also crucial for seed germination. There is a limited and large variation of precipitation, which results in great changes of the soil humidity between different seasons or years in desert, so the moisture is becoming a necessary factor for seed germination [22,23]. Effective water-imbibition of seeds launches seed germination, which is in turn affected by the adjustment of the osmotic pressure generated inside soil and plant [3,24,25]. Seed physiological & biochemical aspects and the osmotic pressure are changed by salt and drought stress, which consequently affect seed germination [2,8,26]. Salt stress can change the integration and permeability restoration of cell membrane, induce the denaturation of proteins in the cell, inhibit DNA synthesis, which has dual function of osmotic stress and ion poisoning [27–30]. Drought stress is usually simulated by polyethylene glycol 6000 (PEG 6000) [31,32]. The previous study showed that effects of isotonic PEG and NaCl on plant was significantly different, which may be caused by the ion regulation mechanism [28,33]. Desert plant seeds often have bracts and winged perianths, e.g. seeds of species in genus of Atriplex, Salsola, Haloxylon [2,12,21,34,35], etc. On the one hand, winged perianth is beneficial for seed dispersal in the desert; On the other hand, it applies certain negative effect in seed germination, which may change with different abiotic factors [34,36,37]. Both of these aspects can effectively adjust the balance between seeds germination at the best time and seeds persistence short or long-term in soil seed bank in early spring [13]. Not only germination but seed vigor and seedling growth can all reflect the effectiveness of germination behavior and the result of stress [2,25]. Abiotic factors, e.g. temperature, salt, drought, etc., impact on plant height, root length of seedlings [10,13]. Previous research showed that a high consistency existed between plant height of early seedling and seed tolerance, rather than root length variation [16,20]. Alive mature seeds will be forced to become dormant or induced into secondary dormancy because of the seed (fruit) structure, e.g. winged perianth, etc. and abiotic stress, e.g. salt and drought, which is one of the adaptive strategy of desert plants in germination [1,2,38, 39]. However, different desert plants evolve different ways to adapt environment [6,22,40], so systematically studying the effect of abiotic factors, e.g. light, temperature, salt, drought, etc., on seed germination and seedling growth should contribute to further understanding of the unique mechanism of the desert plant to adapt to the heterogeneous circumstance in the early stage. Salsola ferganica, an annual desert pioneer halophyte of Salsola genus in Chenopodiaceae family, referred as “natural desalter” [29,41]. Through long-term observation, we found that S. ferganica seeds which have membranous winged perianth could germinate under dense salt crust in early spring when snow
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was melting. It suggests that seed and seedling of S. ferganica are of strong stress tolerance, but the systematic and thorough study of the stress effects on the seed germination and seedling growth is limited. Based on the above situation, in the present study, we investigated the effects of different stress conditions, such as light, storage temperature, day/night temperature variation, salt (NaCl) and drought (PEG 6000), etc. on seed germination (SG) and seedling growth (SGr) of S. ferganica, which may facilitate to further explore the mechanism and adaption strategy of desert plants in early stage, and provide scientific evidence for ecological restoration and reconstruction of the natural environment. 2. Materials and methods 2.1. Seed collection Salsola ferganica usually germinates in late March and early April, blooms in middle of July and August, bears seeds in late September and early October in the natural habitat in Junggar Basin, Xinjiang, China. Seeds of S. ferganica in the present study were collected in October 2013 at Wujiaqu 103 regiment (44°29′821″ N, 87°31′181″ E; 429 m H), Xinjiang. Harvested seeds were air-dried for 2 weeks under room conditions: 18–26 °C, 8–20% relative humidity, then cleaned to remove the impurities and stored in a dry cardboard case in room temperature; meantime, part of seeds were stored at 4 °C. In the present study, seed with membranous appendage (like wing) is named as seed with winged perianth (WWP); seed removed membranous appendages is named as seed without winged perianth (NWP). 2.2. Seed germination experiments Mature intact seeds (WWP, the radius is 3.0–4.5 mm) stored at room temperature or 4 °C were used in seed germination experiments. Four replicates with 30 seeds of each were placed on two layers of filter paper in 9-cm Petri dishes, in which 7 mL of distilled water were added. All Petri dishes were sealed with cling film, and placed in an illuminated incubator (RXZ-500D-LED; Jiangnan Apparatus Manufactory, China), with a 12 h light/12 h dark photoperiod (light flux: approx.100 μmol m−2 s−1), 25 °C, 50% relative humidity. For incubation in darkness, seeds were prepared in the dark room and packaged with foil paper, and checked for germination only at the end of the experiment to avoid exposure to any light during germination. It was considered to be germinated as the radicle of a seed was at least half length of seed without winged perianth. Seed germination was recorded every 24 h for 15 d. The height and root length of seedlings were examined on the 15th day and calculated by Image J 1.47 V (Wayne Rasband National Institutes of Health, USA) with 10 plants in each treatment. Seed germination percentage (G) was calculated as: G = Gi/ Gt × 100%, where Gi represents the number of germinated seeds 15 d later; Gt represents the total number of seeds of each replicate. Seed germination rate (T50/d) is the time (day) that germination percentage reaches the half of the final germination percentage in light [42,43]. 2.3. Test for seed vigor with 2,3,5-triphenyltetrazolium chloride (TTC) After soaking in sterilized distilled water at 28 °C for 20 h, S. ferganica seeds were stained with 0.1% TTC [0.1 g TTC dissolved in 100 mL of 0.1 mol L−1 phosphate buffer (PBS, pH 7.0)] for 12 h at 30 °C in an incubator (EYELA NDO-400, Tokyo Rikakikai C., LTD in Shanghai) in darkness. After being rinsed with distilled water for three times, the treated seeds were examined under stereomicroscope to check the embryo vigor. Seed vigor percentage was calculated according to Zhang and Zhai (2003) [44]. Seeds with stronger vigor were stained in dark red, weaker vigor in pink, inactive seeds would not be stained. Four replicates with 30 seeds of each were applied for one treatment.
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2.4. Seed germination experiments 2.4.1. Light treatment To determine the effect of light on seed germination, white light (wavelength 380–750 nm) and darkness were used in experiment. For seeds in darkness, the Petri dishes were wrapped with foil paper compared to those in light only sealed with cling film [45]. 2.4.2. Day/night temperature variation (D/NTV) treatment Variations of day/night temperature regimes [5 °C in night/15 °C in day (mimic early spring daily temperature regime in natural habitat), 10 °C/20 °C (spring with lower temperature), 15 °C/25 °C (spring with higher temperature), 20 °C/30 °C(late spring)] were applied in seed germination in an illuminated incubator (RXZ-500D-LED; Jiangnan Apparatus Manufactory, China), and subjected to a 12 h in dark (lower temperature)/12 h in light (higher temperature) photoperiod. 2.4.3. Salt and drought treatment Different concentrations of NaCl (0, 50, 100, 300, 500, 700 and 1000 mmol L− 1, corresponding to osmotic pressure − 248, − 496, − 1487, − 2478, − 3469, − 4955 kPa respectively) were applied in seed germination [46]. For PEG treatment, different concentrations of PEG 6000 solution (0, 25, 50, 100, 150, 200 and 300 g L−1, corresponding to osmotic pressure −20, −50, −150, −300, −500, −1000 kPa) were used in seed germination [31]. Sterilized distilled water (osmotic pressure 0 kPa) treatment was used as control. 2.5. Statistical analysis All data were expressed as mean ± standard deviation. One- or twoway ANOVA was used to analyze variable temperature, NaCl and PEG treatment on seed germination and seedling growth, as well as variable temperature on germination rate with the GraphPad Prism Version 5.01 for Windows (GraphPad Software, SanDiego, CA, USA). If significant main effects existed, differences were tested by a multiple comparison Tukey test at 0.05, 0.01, 0.001 significance level. Three-way ANOVA was used to analyze seed type, light, storage time and their interaction
on seed germination of S. ferganica with the SPSS Version 17.0 for Windows (SPSS Inc., Chicago, IL, USA). 3. Results 3.1. Effect of storage time (month) on seed germination and seed vigor 3.1.1. Seed germination The germination trend of S. ferganica seeds at room temperature (RT) and 4 °C storage showed that regardless in light or darkness, seeds without winged parianth (NWP) had high germination percentage with the time increasing; whereas the germination of seeds with winged parianth (WWP) significantly decreased in darkness, which suggests seeds of S. ferganica need light in germination. Besides, germination percentage of NWP seeds was significantly higher than that of WWP seeds at the same storage condition, especially in darkness (Fig. 1). At RT, the germination of NWP seeds and WWP seeds gradually increased and reached the highest percentage in October and kept stable then (Fig. 1A,B); at 4 °C, before November, the germination of S. ferganica seeds was high, while decreased gradually after (Fig. 1C,D). Under light, NWP rather than WWP seeds kept stably higher germination percentage at RT after October (Fig. 1A). NWP seeds showed decreased germination after October; whereas WWP seeds kept higher germination percentage before Feburary of next year and decreased gradually after (Fig. 1C). In darkness, NWP seeds showed similar germination trend under the same storage temperature; whereas WWP seeds had similar germination trend under different storage temperature (Fig. 1B,D). Our results suggest that light applies significant effect to S. ferganica seeds; low temperature delays germination to some extent. Three-way ANOVA analysis (Table 1) of seed type, light, storage time and their interaction on seed germination at different temperatures of S. ferganica showed that regardless at RT or 4 °C, seed types or light types had significant interaction (P b 0.001), for storage time (month), only 4 °C showed significant interaction (P = 0.095 for RT; P b 0.05 for 4 °C); significant interaction existed between seed types and light types both at RT and 4 °C (P b 0.001), while between seed types and
Fig. 1. Change of germination percentage of S. ferganica seeds stored at different temperatures from 2014 to 2015. (A, B) Room tempr. & in light/dark: seeds were stored under room temperature and germinated in light/dark; (C, D) 4 °C & in light/dark: seeds were stored under 4 °C and germinated in light/dark. tempr.: abbreviation of temperature.
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Table 1 Three-way ANOVA analysis of seed type, light, storage time and their interaction on seed germination at different temperatures of S. ferganica. Source of variation
Seed type (S) Light (L) Storage time (Month, M) S ×L S ×M L ×M S ×L ×M
Room temperature
4 °C
d.f.
SS
MS
F-value
P-value
d.f.
SS
MS
F-value
P-value
1 1 12 1 12 12 12
19.352 13.613 0.652 10.199 0.844 0.478 0.676
19.352 13.613 0.054 10.199 0.070 0.040 0.056
571.401 401.950 1.605 301.145 2.076 1.177 1.663
b0.001 b0.001 0.095 b0.001 0.021 0.304 0.080
1 1 12 1 12 12 12
11.855 12.404 1.341 7.714 0.980 0.853 0.957
11.855 12.404 0.112 7.714 0.082 0.071 0.080
212.743 222.600 2.005 138.427 1.466 1.276 1.431
b0.001 b0.001 0.027 b0.001 0.143 0.238 0.157
storage time (month), significant interaction only existed at RT (P = 0.021 for RT; P = 0.143 for 4 °C), no significant interaction was observed between light types and storage time (month) (P = 0.304 for RT; P = 0.238 for 4 °C); no significant interaction existed among seed types, light types and storage time (month) at RT or 4 °C (P = 0.08 for RT; P = 0.157 for 4 °C). Analysis of germination rate of WWP and NWP seeds under varying storage temperatures showed that germination rate of WWP seeds was first increased and then gradually decreased, however, fluctuation existed between different months (Fig. 2). Germination rate of NWP seeds kept stably higher trend. 3.1.2. Seed vigor TTC (2,3,5-triphenyl-2H-tetrazolium chloride) staining can exhibit the status of seed vigor, vigorous seeds are stained with dark red color, lower vigor seeds are stained with pink color, inactive seeds cannot be stained (Fig. 3). S. ferganica seeds were stained with TTC from Oct. 2013, which showed a lower vigor of the newly-mature seeds, and then gradually increased and reached the highest in Apr.–May (spring) of the next year, which showed the completion of after-ripening of seeds (Fig. 4). With the temperature rising, seed vigor presented decrease trend, and had a significant reduction in Jun. of the next year, which may be related to the summer dormancy under high temperature. From Nov. 2014 to Mar. 2015, seed vigor gradually decreased. A correlationship existed between seed vigor monthly change (Fig. 4) to seed germination (Fig. 1). 3.2. Effect of day/night temperature variation (D/NTV) on seed germination and seedling growth 3.2.1. Seed germination D/NTV had little effect on NWP seed germination of S. ferganica, while applied significant effect to that of WWP seeds (Fig. 5). In light (Fig. 5A), NWP seeds at 5 °C day/15 °C night, 10 °C/20 °C, 15 °C/25 °C, 20 °C/30 °C showed high germination percentage, which was more than 95%; while germination of WWP seeds was promoted under
Fig. 2. Change of germination rate between months of S. ferganica seeds stored at different temperatures from 2014 to 2015. Room tempr. & NWP/WWP: seeds without/with winged perianth were stored at room temperature; 4 °C & NWP/WWP: seeds without/with winged perianth were stored at 4 °C. tempr.: abbreviation of temperature.
various D/NTV (≥ 85% compared to the control 80%) except for 5 °C/15 °C (about 45%). In darkness (Fig. 5B), there had little effect of D/NTV on NWP seed germination (≥ 95%) except for 20 °C/30 °C (about 70%); WWP seed germination was significantly promoted at 5 °C/15 °C and 10 °C/20 °C D/NTV (about 67% and 80%) compared to control (17%), while no significant effect was at 15 °C/25 °C and 20 °C/30 °C. As shown in Fig. 5, although lower D/NTV significantly affected seed germination of S. ferganica in light, it could partially counteract the inhibition effect of darkness on seed germination. Analysis of germination rate under D/NTV showed that WWP seed germination rate increased with the rising of D/NTV (Fig. 6), germination at 5 °C/15 °C was the slowest, 20 °C/30 °C was the fastest, D/NTV had little effect on germination rate of NWP seeds. 3.2.2. Seedling growth Analysis of seedling growth of S. ferganica under D/NTV showed that, regardless in light or darkness, lower D/NTV (5 °C/15 °C) promoted NWP and WWP seedling growth (8.686 mm and 8.768 mm for height; 6.233 mm and 5.744 mm for root length) (Fig. 7A,C); NWP seedling growth reduced with the rising of D/NTV. In light, NWP seedling growth was better than that of WWP, especially at 15 °C/25 °C. In darkness, WWP seedling growth was the best at 5 °C/15 °C, while NWP seedling growth was promoted at various D/NTV (especially at 10 °C/20 °C) except for 20 °C/30 °C (Fig. 7B,D). NWP seedling growth was better in darkness than that in light. 3.3. Effect of salinity or drought on seed germination and seedling growth Under lower NaCl concentration [100 mmol L−1, −496 kPa osmotic pressure (OP)], WWP seed germination had no significant difference compared to control (Fig. 8A); under middle NaCl concentration (300 mmol L−1, − 1487 kPa OP), seed germination reduced to about 50%; under higher NaCl concentration (500 mmol L−1, − 2478 kPa OP), seed germination remained about 40%, which suggests S. ferganica can tolerate higher salinity. Under lower concentration of PEG (b150 g L− 1, ≥− 300 kPa OP), WWP seed germination had no significant difference compared to control (Fig. 8B); under middle concentration (200 g L−1, −500 kPa OP), seed germination remained more than 60%; under higher concentration (300 g L−1, −1000 kPa OP), seed germination decreased significantly. Although 100 mmol L−1 NaCl and 200 g L−1 PEG had nearly the same
Fig. 3. TTC staining of S. ferganica seeds. (A) Dark red seeds; (B) pink seeds; (C) unstained seeds.
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Fig. 4. The vigor of S. ferganica seeds stored at room temperature by TTC method from 2014 to 2015. Rs: dark red seeds (stronger vigor); Ps: pink seeds (weaker vigor); Us: unstained seeds (inactive vigor).
OP, seed germination and seedling growth of the former were significantly greater than that of the latter. Analysis of seedling growth of S. ferganica showed that effect of NaCl or PEG treatment was greater on NWP than that of WWP seedling growth, especially PEG (Fig. 9). With the rising of NaCl and PEG concentration, both NWP and WWP seedling height and root length were inhibited (Fig. 9A,B,C,D). Under similar OP of NaCl or PEG treatment, PEG had greater effect on NWP seedling growth while had little effect on that of NWP than that of NaCl. 4. Discussion The adaptability of seed germination and seedling growth is pivotal for plant establishment, succession of the population and propagation of species to the heterogeneous environment. In the present study, we investigated the effects of different stresses on seed germination (SG) and seedling growth (SGr) of S. ferganica, an annual halophyte in desert, which may facilitate to explore the mechanism and adaptation strategy in early stage of plant growth. Our results showed that light quality, day/ night temperature variation, and winged perianth etc., applied significant effects on SG and SGr; S. ferganica SG and SGr could tolerate high salinity and drought. 4.1. Seed germination of S. ferganica was sensitive to light Desert halophytes show different sensitivity to light in seed germination. For some plant species, light has crucial function on SG and SGr [7,46,47], e.g. Cyperus arenarius [8] and Cyperus conglomeratus [18]
Fig. 6. Effect of day/night temperature variation (D/NTV) on germination rate of S. ferganica in light. Lowercase letters are variance analysis results of germination rate, different letters show significant difference (P b 0.05). *, **, *** indicate significant difference by t-test (P b 0.05, 0.01, 0.001). Horizontal ordinate represents D/NTV: 25 °C, germination at 25 °C for 24 h; 5 °C/15 °C, germination at 5 °C for 12 h in darkness and at 15 °C for 12 h in light. The sets of other D/NTV were similar with 5 °C/15 °C.
in Cyperaceae family, Sporobolus ioclados [48] (Gramineae), Tamarix aucheriana [49] (Tamaricaceae), Halostachys caspica [15] (Chenopodiaceae), etc., which can achieve much better germination in light compared to that in darkness. In our present study, germination of S. ferganica seeds with winged perianth was sensitive to light, seed germination was much higher in light than in darkness under the same treatment. On the contrary, the germination of some desert species, e.g. Panicum turgidum [17] (Gramineae), Tanacetum cinerariifolium [16] (Compositae), etc., get the highest germination percentage in darkness [16–18], which suggests the characteristics of light independence [47]. Still some other species, e.g. Suaeda salsa [20,50], Suaeda aralocaspica [10,11] and Salsola affinis [21] in Chenopodiaceae etc., present similar seed germination behavior in light or darkness, which is insensitive to light in germination. Light applies effect on both seed germination and early seedling growth, however, they might have completely different responses in light requirement and light sensitivity [47]. Requirement of different light wavelength in seed germination and seedling photomorphogenesis is probably related to different adaptation strategies of various desert plant species [13,43]. In our study, the seedling of S. ferganica grew better in darkness than that in light, which suggests that darkness may promote hypocotyl elongation and radicle protrusion at early stage of germination. The effects of light with different wavelength on SG and SGr depend on plant species and genetic factors [47,51], e.g. seed germination of Lactuca sativa is red light-dependent [52]. The previous research showed that seed germination affected by different light qualities may be related to the phytochromes [53,54].
Fig. 5. Effect of variable temperature on germination of S. ferganica in light (A) and dark (B). Lowercase letters are variance analysis results of seed germination, different letters show significant difference (P b 0.05). Horizontal ordinate, daily temperature regime: 25 °C, seed germination temperature kept at 25 °C for 24 h; 5/15 °C, seed germination temperature kept at 5 °C in dark for 12 h, and kept at 15 °C in light. The sets of other daily temperature regimes were similar with 5/15 °C.
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Fig. 7. Effect of D/NTV on seedling growth of S. ferganica in light (A,B) and darkness (C,D). Capital and lowercase letters are variance analysis results of seedling height and root length, different letters show significant difference (P b 0.05). *, **, *** indicate significant difference by t-test (P b 0.05, 0.01, 0.001). Horizontal ordinate represents D/NTV: 25 °C, germination at 25 °C for 24 h; 5 °C/15 °C, germination at 5 °C for 12 h in darkness and at 15 °C for 12 h in light. The sets of other D/NTV were similar with 5 °C/15 °C.
4.2. Appropriate temperature and its variation were important for seed germination and seedling growth of S. ferganica Temperature as one of the important factor of environmental stress plays vital role for seed germination and early seedling growth [13,38]. Temperature fluctuates largely among different years, and shows drastic change between day and night, which is especially true in early spring as snow melting in desert habitat [6,23]. The desert plant can respond to temperature variation and germinate rapidly [2,22,38]. Some desert plant can germinate in a wide temperature range, Hordeum jubatum [55] (Gramineae) and Haloxylon stocksii [56] (Chenopodiaceae) have the highest germination percentage at a day/night temperature variation of 5 °C/20 °C and 10 °C/30 °C respectively. In the present study, S. ferganica showed the optimum germination at 10 °C/20 °C. The desert plants such as Suaeda physophora [57], Atriplex stocksii [58], Salsola affinis [8,21] all have the highest germination percentage at a day/night temperature variation of 10 °C/20 °C. Similar range of the optimum germination temperature of plants which lived in similar habitat is a characteristic of convergence, in which plant germinates in mid April when temperature is rising (corresponding to the optimum germination range), suggesting the adaptability of these plant species to early spring weather [2,7,13,38].
Seed germination can significantly be affected by changes of environmental temperature in seed storage. In the present study, S. ferganica seeds showed reduced germination percentage in June–August, which may be in connection with summer dormancy of this species under high temperature [59]. Poor germination of Limonium stocksii [60] (Plumbaginaceae) and Atriplex centralasiatica [61] (Chenopodiaceae) was observed at high day/night temperature range of 25 °C/35 °C. Seed vigor can be significantly influenced by storage time, storage temperature and internal components of seed itself [7,62,63]. Seed vigor of Haloxylon (Chenopodiaceae) dramatically decreased after 9–12 months storage at room temperature, and poor germination was present after 17 months [15]. In our experiment, S. ferganica seed remained high seed vigor (germination more than 90%) after 13–15 months storage, which suggests that S. ferganica seeds can keep vigor for longer. 4.3. Seed accessory structure applied special ecological role in seed germination of S. ferganica Desert plant species usually develop accessory structures with seeds to acclimate environment. The winged perianth of seed has an important role in ecological adaptability [7,36]. It can contribute to seed dispersal, and force seeds into secondary dormancy [37,59]. However, as
Fig. 8. Effect of NaCl or PEG on germination of S. ferganica. Lowercase letters are variance analysis results of NaCl or PEG on germination, different letters show significant difference (P b 0.05). *, **, *** indicate significant difference by t-test (P b 0.05, 0.01, 0.001).
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Fig. 9. Effect of NaCl (A,C) or PEG (B,D) on seedling growth of S. ferganica in light (A,B) and darkness (C,D). Capital and lowercase letters are variance analysis results of seedling height and root length, different letters show significant difference (P b 0.05). *, **, *** indicate significant difference by t-test (P b 0.05, 0.01, 0.001).
the natural mechanical barrier between seed and its original surrounding, winged perianth can assist desert seeds to choose optimum chance to germination, which fits for “bet-hedging or conservative strategy” of seed germination [2,7,8,13,38]. In the present study, S. ferganica germination percentage of NWP seeds was higher than WWP seed under the same condition. A number of desert plant seeds with winged perianth or bract are of such characteristics [12,21,36,64]. Probably NWP seeds can quickly respond to light without mechanical block of winged perianth and germinate rapidly. In addition, endogenous hormones or chemicals in winged perianth may apply effect on seed germination [2,59]. 4.4. Seed germination and seedling growth of S. ferganica were tolerant to salt and drought stress Whether desert halophyte can survive in saline environment or not depends on seed and seedling responses to salt and drought stress [2,26]. Salt tolerance is much different among halophytes. Seed germination of Suaeda aralocaspica was more than 10% at 1400 mmol L−1 NaCl [10,11]; whereas that of Lepidium latifolium (Cruciferae) [65] and Limonium iconicum [66] (Plumbaginaceae) is almost 0 at 300 mmol L−1 NaCl. In our study, S. ferganica could germinate more than 3% at 700 mmol L−1 NaCl, which suggests that S. ferganica is relatively salt-tolerant. For desert halophytes, lower NaCl concentration can promote seed germination, however, higher NaCl concentration may force them into secondary seed dormancy [7,13,59]. Halophytes in Haloxylon genus [15,56], Suaeda genus [10,11,20,50], Salsola genus [12,21] (including S. ferganica in the present study) present similar salt tolerance in seed germination. Besides, our results showed that S. ferganica was drought-tolerant, which had similar trend with salt tolerance, while seed germination and seedling growth under NaCl treatment were significantly better than that of PEG treatment in which they shared the same osmotic pressure. It suggests that salt ions may relieve the effect of osmotic stress [27,28]. Our results agreed with the previous report which showed that the stress effect of PEG was significantly greater than that of NaCl [33]. 5. Conclusion Abiotic factors e.g. salt, drought, day/night temperature variation, and intense light irradiation etc., seriously influence desert plant
growth, especially in the critical periods of seed germination and seedling growth. Germination behavior and seed dormancy strategy have great effect on whole life of plants, which in turn impact population establishment and species propagation [2,7,8,13,38,59]. Our results showed that S. ferganica needed light in seed germination; the winged perianth applied certain negative effect in germination, while it also could adjust the balance between seed germination and dormancy in the proper time; appropriate day/night temperature variation promoted S. ferganica seed germination, while low temperature delayed germination process in some extent; seed germination and seed vigor of S. ferganica gradually decreased with the storage time increasing; seeds and seedlings of S. ferganica were salt- and drought-tolerant. Based on the above result, we speculate that S. ferganica employs ‘cautious strategy’ in germination, which means that under favorable conditions of light, temperature, water, etc., S. ferganica can germinate actively and get enough seedlings developing into adult plants; whereas under unfavorable conditions, seeds may not germinate or germinate in a small amount to replenish soil seed bank [2,7]. Acknowledgements This work was supported by the Project for Scientific Research Project in higher education of Xinjiang Uygur Autonomous Region (XJEDU2014S084); National Natural Science Foundation of China (31060027; 31260037; 31460043); Project for Training Young Talents of Xinjiang Uygur Autonomous Region (2013721013). References [1] Y. Zhang, L.G. Xue, T.P. Gao, L. Jin, L.Z. An, Research advance on seed germination of desert plants, J. Desert Res. 25 (1) (2005) 106–112. [2] X.X. Qu, Z.Y. Huang, The adaptive strategies of halophyte seed germination, Acta Ecol. Sin. 25 (9) (2005) 2389–2398. [3] L. Rajjou, M. Duval, K. Gallardo, J. Catusse, J. Bally, C. Job, D. Job, Seed germination and vigor, Annu. Rev. Plant Biol. 63 (3) (2012) 507–533. [4] Q. Sun, J.H. Wang, B.Q. Sun, Advances on seed vigor physiological and genetic mechanisms, Sci. Agric. Sin. 40 (1) (2007) 48–53. [5] Z.H. Li, J.H. Wang, Advances in research of physiological and molecular mechanism in seed vigor and germination, Sci. Agric. Sin. 48 (4) (2015) 646–660. [6] C.C. Baskin, J.M. Baskin, Germination ecophysiology of herbaceous plant species in a temperate region, Am. J. Bot. 75 (2) (1988) 286–305. [7] C.C. Baskin, J.M. Baskin, Seeds: Ecology, Biogeography and Evolution of Dormancy and Germination, Academic Press, San Diego, 1998.
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