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Published online 19 March 2008
Published in Crop Sci 48:756-762 (2008)
© 2008 Crop Science Society of America
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Improving Seed Germination of Saltgrass under Saline Conditions

M. A. Shahbaa, Y. L. Qiana,* and K. D. Lairb

a Dep. of Horticulture and Landscape Architecture, Colorado State Univ., Fort Collins, CO 80523-1173
b Bureau of Reclamation, Technical Service Center, Denver, CO 80225. The research was, in part, supported by the U.S. Golf Association and the U.S. Department of the Interior, the Bureau of Reclamation, through the Rocky Mountains Cooperative Ecosystem Studies Unit

* Corresponding author (Yaling.Qian{at}Colostate.edu).


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Saltgrass [Distichlis spicata var. stricta (Greene)] has a great potential for use as a turfgrass and as a revegetation species of saline sites. Experiments were conducted to test the effect of the application of different concentrations of ethephon, fusicoccin, kinetin, thiourea, and Proxy on saltgrass seed germination under three salinity levels. Saltgrass germination percentage was 56% under nonsaline condition, which was reduced to 46 and 26% at 15 and 30 dS m–1 salinity levels, respectively. Ethephon application (5 mM) increased saltgrass germination percentage under the highest salinity treatment (30 dS m–1) only. However, Proxy (at 5 mM a.i.) increased saltgrass germination under all salinity treatments, reaching 97, 76, and 40% under control, 15 dS m–1, and 30 dS m–1 salinity levels, respectively. Kinetin at 0.5 to 1.0 mM did not increase saltgrass germination under nonsaline conditions but increased germination percentage by 35% at 15 dS m–1 and by 89% at 30 dS m–1 salinity. Fusicoccin (at 10 µM) and thiourea (at 30.0 mM) also increased germination percentage under all salinity treatments. Our investigation showed that 5.0 mM ethephon, 10 µM fusicoccin, 0.5 to 1.0 mM kinetin, 30 mM thiourea, and Proxy (at 5 mM a.i.) increased saltgrass seed germination under saline conditions. Proxy was the most effective in improving saltgrass germination percentage under saline conditions, followed by thiourea, fusicoccin, ethephon, and kinetin.

Abbreviations: ABA, abscisic acid • EC, electrical conductivity


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
SALTGRASS [Distichlis spicata var. stricta (L.) Greene], native to the western United States, has desirable turf characteristics that include fine texture, good color, and high shoot density. Its biological attributes include tolerance to wear, compaction, drought, and salinity (Fraser and Anderson, 1980; Kopec and Marcum, 2001; Qian et al., 2007). Saltgrass has been classified as a halophyte (O'Leary and Glenn, 1994); it has a great potential for use as a turfgrass of saline sites.

Saltgrass has also been used in revegetation mixtures with alkali sacaton [Sporobolus airoides (Torr.) Torr.] to restore mesic or subirrigated saline meadows and riparian sites in the western United States (Lair and Wynn, 2002a, 2002b).

Revegetation by direct seeding could be more effective than planting rhizomes. Establishment and revegetation using saltgrass rhizomes is labor intensive with specialized requirements. Establishment by direct seeding could be more efficient than planting rhizomes (Cluff and Roundy, 1988). However, despite their great salt tolerance after establishment, the germination of many halophytes (including saltgrass) could be inhibited under saline conditions. Previously, seeds of saltgrass accessions were evaluated for their germination ability under different levels of salinity. It was found that saltgrass germination was significantly reduced when salinity exceeded 8 to 12 dS m–1 (Christensen and Qian, 2004). Saltgrass appears to be more sensitive to salinity during germination than established turf stands (Qian et al., 2007). The inhibitory effect of salinity to saltgrass germination included reducing germination percentage, delaying the germination process, and causing a loss of seed viability.

The effects of germination regulating chemicals in enhancing seed germination under salinity conditions and alleviating salinity stress has been reported in several plant species. Gul and Khan (2003) demonstrated that a substantial enhancement in seed germination of the perennial halophyte Utah pickleweed (Salicornia utahensis Tidestrom) with the inclusion of ethephon (10 mM) and kinetin (0.05 mM). Ethephon is an ethylene-releasing compound. Ethylene may stimulate seed germination (Whitehead and Nelson, 1992; Sutcliff and Whitehead, 1995), especially when seeds are exposed to salt and temperature stresses (Khan and Andreoli, 1993; Li et al., 1995). El-Keblawy et al. (2005) reported a positive effect of 0.05 mM kinetin in enhancing the germination at high salinity levels in mesquite [Prosopis juliflora (Sw.) DC.]. Khan and Ungar (2001a) reported that thiourea (10 mM) partially alleviated the inhibitory effects of salinity on the germination of summer seeds of coastal dune grass [Halopyrum mucronatum (L.) Stapf], while kinetin (0.05 mM) alleviated the inhibitory effects of salinity on the germination of winter seeds. Fusicoccin is known to alleviate the effect of salinity on the seed germination of halophytes (Ismail, 1990; Gul and Weber, 1998; Gul et al., 2000; Khan et al., 2002; Gul and Khan, 2003).

The objectives of this study were (i) to determine whether applications of ethephon, fusicoccin, kinetin, thiourea, or a commercial ethephon product, Proxy (Bayer Environmental Science, Montvale, NJ), could promote saltgrass seed germination under different salinity levels and (ii) to determine the most effective concentrations of each growth regulator in enhancing saltgrass germination under saline conditions.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Saltgrass inflorescences were collected in August 2005 from a saltgrass nursery at the Colorado State University Horticulture Research Farm, Fort Collins, CO. The inflorescences represented a composite of different saltgrass accessions. Seeds were obtained by thrashing inflorescences against a rough vinyl surface. Seeds were then further separated from hulls and other debris using a seed blower (757-RC South Dakota Seed Blower, Seedburo Equip., Chicago, IL). Seeds were stored at room temperature before starting the studies.

Without pretreatment, saltgrass seeds have a low germination due to seed dormancy. Previously, Qian et al. (2006) found that stratification and machine scarification improved germination of saltgrass seeds. To break seed dormancy, seeds were subjected to machine scarification. Scarification was performed by setting a scarifier (MAT-OSU pneumatic seed scarifier, Mater Intel., Corvallis, OR) at 112 MPa pressure and 60-grit sandpaper was used for 4 min (Qian et al., 2006).

After seed scarification, three treatment factors were imposed: salinity, type of germination-regulating chemical, and concentration of germination-regulating chemical. The experiment was set up in the growth chamber and repeated once. Split-split plot design with three replications was used. Salinity levels were considered as the whole plot factor, germination-regulating chemicals were the subplot factor, and the concentrations of these chemicals were the sub-subplot factor. Salinity levels were control (distilled water), electrical conductivity (EC) = 15, and 30 dS m–1 prepared using NaCl as the germination solution. Chemicals used were ethephon, fusicoccin, kinetin, thiourea, and Proxy, a commercial source of ethephon which contains 21.7% of ethephon and 78.3% of other inert and/or unknown materials. Four different concentrations of each were used: ethephon (0.0, 5, 10, and 20 mM), fusicoccin (0.0, 5.0, 10.0, and 20.0 µM), kinetin (0.0, 0.5, 1.0, 1.5 mM), thiourea (0.0, 10, 20, and 30 mM), and Proxy (four a.i. ethephon concentrations: 0.0, 5.0, 10.0, and 20.0 mM). All chemicals were prepared by using NaCl solutions at 0, 15, and 30 dS m–1, respectively, depending on salinity treatment levels.

Seeds were sown on sterile germination blotter papers lined in 9-cm diameter petri dishes. In each dish, 40 seeds were placed on each germination blot. Germination blots were moistened with 10 mL of different treatment solutions. Petri dishes were then sealed with parafilm and were placed in a growth chamber (Model MB-60B, Percival Manufacturing, Boone, IA). The growth chamber was programmed to maintain alternating 16 h warm/8 h cool temperatures of 35/15°C in each 24-h period according to Cluff and Roundy (1988) who found these conditions are optimal for saltgrass germination. A photoperiod of 16 h light/8 h dark (350 µmol m–2 s–1 of photosynthetically active radiation), concurrent with warm/cool temperature periods, was provided by fluorescent and incandescent lights.

Germination was recorded every other day after 2 d for 20 d. Seeds were considered germinated if the emerged radical was visible. Germination percentage was defined as the total percent germination in 20 d. The speed of germination was calculated by dividing the percentage of seeds germinated at each count by the number in days from the start of the germination test. The total of values obtained is the germination speed (Maguire, 1962).

Data Analysis
The data of the two experiments were subjected to ANOVA to test the experiment effect and the interaction between treatments and experiments. The experimental run was not significant. Therefore, data were pooled over experiments to test the effects of salinity levels and different concentrations of each chemical treatment on germination speed and percentage at individual salinity levels (SAS Institute, 2005). Means separation were performed at P = 0.05 by Fisher's LSD test when significant differences were found. The most effective concentration of each chemical treatment was chosen and subjected to ANOVA for chemical treatment comparison.


   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Analysis of variance indicated significant salinity, chemical type, and concentration effects on saltgrass germination percent and speed. Also, the interactions between chemical type and concentration were significant (Table 1 ).


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  		Table 1. Analysis of variance with mean squares and treatment significance of saltgrass seed germination speed (% d–1) and percentage as affected by different chemical treatments, chemical concentrations, and their interaction.

 
The increase in salinity significantly reduced the germination percentage and speed of saltgrass (Fig. 1 ). Saltgrass germination percentage was 56% under nonsaline condition, which was reduced to 46 and 26% at 15 and 30 dS m–1 salinity levels, respectively. Germination speed was reduced from 20.4% d–1 under nonsaline conditions to 16.5 and 8.8% d–1 at 15 and 30 dS m–1 EC, respectively (Fig. 2 ). Effects of NaCl on seed germination could be osmotic and/or ionic either by reducing water availability, interfering with some aspect of metabolism, or altering the balance of the endogenous growth regulators in the seeds (Khan and Ungar, 1998a, 1998b; Khan and Ungar, 2001c; Ungar, 1991).


Figure 1
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  		Figure 1. Effect of different concentrations of ethephon, fusicoccin, kinetin, thiourea, and Proxy on saltgrass seed germination percentage under different salinity levels. Columns labeled with different letters are significantly different at P = 0.05 within each salinity level.

 

Figure 2
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  		Figure 2. Effect of different concentrations of ethephon, fusicoccin, kinetin, thiourea, and Proxy on saltgrass seed germination speed under different salinity levels. Columns labeled with different letters are significantly different at P = 0.05 within each salinity level.

 
Ethephon in the form of 2-chloroethyl phosphonic acid, significantly improved germination percentage (Fig. 1) at EC = 15 and 30 dS m–1 but only improved germination speed at EC = 30 dS m–1 (Fig. 2). Ethephon treatment did not significantly enhance germination under nonsaline conditions. Ethephon at 5.0 mM was the best concentration in relieving the inhibitory effects of salinity on saltgrass germination percentage and speed. It increased germination percentage at EC = 15 dS m–1 by 29.1% and at EC = 30 dS m–1 by 116.1% compared to the respective controls. Higher concentrations of ethephon (10.0 and 20.0 mM) adversely affected both germination percentage and speed. In agreement with our findings, several previous studies indicated the role of ethephon in reducing dormancy in the seeds of several species and reversing the inhibitory effect of abscisic acid (ABA) and osmotic stress (Schonbeck and Egley, 1981; Gulzar and Khan, 2002; Gul and Khan, 2003). Ethephon significantly ameliorated the effect of salinity, ranging from 36 to 54 dS m–1, in iodine bush [Allenrolfea occidentalis (S. Wats.) Kuntze] (Gul and Weber, 1998), Utah pickleweed (Gul and Khan, 2003), and dropseed (Sporobolus ioclados Nees ex Trin) (Gulzar and Khan, 2002). However, the effectiveness of ethephon in ameliorating salinity-induced dormancy is quite variable among plant species. It partially alleviated salinity-induced dormancy in Arthrocnemum indicum (Wild.) Moq. (Khan et al., 1998) and saltwort (Salicornia rubra A. Nels.) (Khan et al., 2002). In contrast, it had no effect on the seed germination of seaside arrow grass (Triglochin maritima L.) under salinity conditions (Khan and Ungar, 2001c).

Proxy is a commercial product with ethephon as its active ingredient. In this study, Proxy significantly improved germination percentage (Fig. 1) and germination speed (Fig. 2) of saltgrass at all salinity levels. Proxy (at 5 mM a.i.) achieved the best germination under all salinity levels. Proxy (at 5 mM a.i.) increased germination percentage by 86, 90, and 108% under control, 15 dS m–1, and 30 dS m–1 salinity levels, respectively. Proxy was significantly more effective in enhancing saltgrass germination percentage than ethephon at all salinity levels except at EC = 30 dS m–1 (Fig. 3 ). The fact that Proxy was more effective than ethephon could be related to the effect of the other unknown ingredients of Proxy.


Figure 3
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  		Figure 3. Effect of ethephon, fusicoccin, kinetin, thiourea and Proxy (at their optimal concentrations) on (A) saltgrass seed germination percentage and (B) germination speed under different salinity levels. Columns labeled with different letters are significantly different within the same salinity level at P = 0.05.

 
Germination percentage (Fig. 1) and germination speed (Fig. 2) of saltgrass were significantly improved by the presence of fusicoccin in the germination solution on the blotter paper at all salinity levels. Analysis of variance and means separation test indicated that the level of 10.0 µM of fusicoccin was the best level in ameliorating the effect of salinity on saltgrass seed germination at 15 dS m–1 salinity. It increased germination percentage at EC = 15 dS m–1 by 53.9% of the control. The level of 5.0 µM of fusicoccin achieved the highest germination speed and percentage at EC = 30 dS m–1. It increased germination percentage by 115.0% when compared to the control without fusicoccin presence. Fusicoccin was originally identified as a toxic by-product synthesized by the fungus Fusicoccin amygdale Del. Fusicoccin contains three fused carbon rings and another ring which contains an oxygen atom and five carbons. Alleviation of salinity effect on seed germination by fusicoccin was reported in many other halophytes such as Zygophyllum qatarensis Hadidi (Ismail, 1990), Utah pickleweed (Gul and Khan, 2003), and triangle orache (Atriplex prostrata Bouchér ex DC.) (Khan et al., 2003). Conversely, El-Keblawy et al. (2005) reported the failure of fusicoccin to stimulate germination in mesquite. Fusicoccin may stimulate ATPase during the early phases of germination to facilitate proton extrusion and K+ uptake (Marre, 1979). Cocucci et al. (1990) studied the response of radish (Raphanus sativus L.) seeds to osmotic medium and fusicoccin during the early germination stages and indicated that fusicoccin counteracted the inhibitory effect of salinity in the medium by enhancing H+ extrusion and synthesis of malic acid. Lutsenko et al. (2005) suggested that fusicoccin affects the ionic balance, especially the K+/Na+ ratio, aiding in ionic homeostasis in seed and embryo. Salinity stress enhances ABA production, which has inhibitory effect on seed germination. Fusicoccin has the ability to remove the inhibitory effect of ABA on seed germination by accelerating development and by replacing the requirements for light and endogenous hormones in breaking dormancy (Lado et al., 1975).

Kinetin had no significant effect in enhancing saltgrass seed germination under nonsaline conditions. It was effective in improving both germination speed and percentage at EC = 15 and 30 dS m–1. At EC = 15 dS m–1, 1.0 mM of kinetin achieved the highest germination percentage (Fig. 1) and speed (Fig. 2). At EC = 30 dS m–1, 0.5 and 1.0 mM kinetin achieved equal effect on saltgrass seed germination speed (Fig. 2), but the level of 0.5 mM had the highest effect in improving germination percentage (Fig. 1). At EC = 15 dS m–1, 1.0 mM of kinetin increased germination percentage by 43.4% and at EC = 30 dS m–1, 0.5 mM of kinetin increased germination percentage by 99.5% of the control. Kinetin has been demonstrated to ameliorate the salinity-induced germination inhibition in Utah pickleweed (Gul and Khan, 2003), Brassica campestris L. (Ozturk et al., 1993), Zygophyllum simplex L. (Khan and Ungar, 1997), Halopyrum mucronatum (Khan and Ungar, 2001a), and Salicornia rubra (Khan et al., 2002). Kinetin partially ameliorated salinity inhibitory effect on the seed germination of mesquite (El-Keblawy et al., 2005), seaside arrow grass (Khan and Ungar, 2001c), and Aeluropus lagopoides (L.) Trin. ex Thw. (Gulzar and Khan, 2002). However, it had no effect on the salinity-induced dormancy in Sporobolus ioclados Nees ex Trin and Urochondra setulosa Trin (Gulzar and Khan, 2002), Salicornia pacifica Standl. (Khan and Weber, 1986), Zygophyllum qatarense (Ismail, 1990), Sporobolus arabicus Boiss (Khan and Ungar, 2001b), Cressa cretica L., Suaeda fruticosa auct. non Forsk., Salsola imbricate Forssk., and Haloxylon stocksii Boiss. (Gulzar and Khan, 2002; 2003). Khan and Ungar (2001a) suggested that addition of kinetin likely overcomes the deficiency in growth-promoting substances that are inhibited in salt-stressed seeds. The increase in seed germination under high salinity after exogenous application of kinetin was attributed to the ability to enhance water uptake during germination (Sastry and Shekhawat, 2001). Miller (1961) suggested that kinetin effect on the breaking of dormancy and promotion of seed germination may result from its combination of influences on cell division and enlargement. Also, kinetin enhances the biosynthesis of ethylene. As discussed previously, ethylene or ethylene-releasing compounds enhance germination when seeds are exposed to salt stress.

Thiourea significantly improved germination percentage (Fig. 1) and germination speed (Fig. 2) of saltgrass at all salinity levels. Analysis of variance and means separation test indicated that 30.0 mM of thiourea is the most effective concentration in improving both germination speed and percentage at all salinity levels. The level of 30.0 mM of thiourea increased germination percentage at EC = 0.0 dS m–1 by 71%, at EC = 15 dS m–1 by 59%, and at EC = 30 dS m–1 by 104% of the controls. Thiourea concentration of 30.0 mM had a similar trend of effect on germination speed. This result is consistent with previous investigations that had demonstrated the effectiveness of thiourea in ameliorating salinity-induced inhibition of germination. Thiourea significantly alleviated the salinity induced dormancy in summer seeds of Halopyrum mucronatum (Khan and Ungar, 2001a), Sporobolus arabicus (Khan and Ungar, 2001b), Salicornia rubra (Khan et al., 2002), Atriplex prostrata (Khan et al., 2003), Zigophyllum simplex (Khan and Ungar, 1997), Aeluropus lagopoides (Gulzar and Khan, 2002), and Triticum aestivum L. (Siddiqui et al., 2006). Thiourea partially alleviated the germination inhibition under saline conditions in mesquite (El-Keblawy et al., 2005), seaside arrow grass (Khan and Ungar, 2001c), and iodine bush (Gul et al., 2000). The fact that thiourea stimulates seed germination and reduces the negative effects of salinity on germination supports that thiourea is an important compatible osmoregulator (Gul et al., 2000). Eashi et al. (1979) found that nitrogenous compounds such as thiourea could promote germination by acidification and softening of cell walls, or by activating the pentose phosphate pathway. Salinity causes a reduction in growth promotors (cytokinins and gibberellins) and increases in ABA in seeds (Kabar and Baltepe, 1990). Thiourea may counteract this effect, controlling the adverse changes through a balance between hormonal promotors and inhibitors.

Analysis of variance indicated a significant difference among tested chemicals at EC = 0.0 dS m–1 and EC = 15 dS m–1 but there was no significant difference at EC = 30 dS m–1 (Table 1) in their effect on enhancing saltgrass germination percentage (Fig. 3A) and germination speed (Fig. 3B).

Our investigation showed that 5.0 mM ethephon, 10 µM fusicoccin, 0.5mM kinetin, 30 mM thiourea, and Proxy at 5.0 mM a.i. increased seed germination percentage and speed of saltgrass under saline conditions. Proxy was the most effective growth regulator in ameliorating salinity effect on saltgrass seed germination at 15 dS m–1, followed by thiourea, fusicoccin, ethephon, and kinetin. All tested growth regulators had similar positive effects at the highest salinity level (EC = 30 dS m–1). More research is needed to develop appropriate protocols for practical and effective treatment procedures for vegetation restoration of saline areas or establishing turfgrass under saline conditions using saltgrass.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
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Received for publication July 11, 2007.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 





This Article
Right arrow Abstract Freely available
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Right arrow Full Text (PDF) Free
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Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shahba, M. A.
Right arrow Articles by Lair, K. D.
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PubMed
Right arrow Articles by Shahba, M. A.
Right arrow Articles by Lair, K. D.
Agricola
Right arrow Articles by Shahba, M. A.
Right arrow Articles by Lair, K. D.
Related Collections
Right arrow Soil Salinity
Right arrow Turfgrass
Right arrow Seed Physiology

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