Comparison of prechilling stratification and sulfuric acid scarification on seed germination of Panicum virgatum under drought stress

Abstract

In semi-arid regions of the Loess Plateau, water deficiency restricts plant performance. Panicum virgatum (switchgrass), which is a highly versatile grass, had been introduced to the Plateau as a restoration species. To determine if prechilling stratification (PCS) and sulfuric acid scarification (SAS) can optimize establishment,P. virgatum cvs Pathfinder, Trailblazer and Alamo were tested under different ambient water potentials by measuring germination and root and shoot growth along water potential gradients under laboratory conditions. Both PCS and SAS improved total germination percentage (TGP), with PCS being more beneficial. The effect of PCS and SAS on mean germination time (MGT) weakened gradually with increasing drought stress. Both PCS and SAS showed no obvious effect on promoting root and shoot growth. Both PCS and SAS reduced base water potential requirement for reaching 50% germination of Pathfinder and Trailblazer, with this effect greater for PCS. These results indicate that embryo dormancy may be a major factor limiting germination ofP. virgatum under drought conditions. Pathfinder appears to be more suitable for a semi-arid environment, whereas Alamo appears to be unsuitable for drought conditions. Given the large difference between predicted value and measured value, the reliability and applicable scope of linear regression estimatedY₅₀ needs further investigation, specification and optimization.

Keywords

base water potential / data analysis method / embryo growth / germination

1 Introduction

Panicum virgatum (switchgrass), native to North America, is a highly versatile grass, used for ecology restoration, livestock forage feed and bioenergy^[1^]. In recent years, switchgrass has been introduced as a restoration species on the Loess Plateau of China^[2^], where habitat destruction has increased due to the limited and erratic precipitation. Furthermore, the Loess Plateau is considered to have potential for bioenergy production using plants also useful as livestock forage^[3^]. Thus, increasing attention is being given to the cultivation of switchgrass on the Loess Plateau^[4^].

Although switchgrass has been widely adopted, seed dormancy is a major factor limiting plant establishment, causing delayed and sporadic emergence, limiting the benefits of commercial adoption^[5^]. Research has focused on methods to effectively break switchgrass dormancy^[6,7^], and has shown that breaking both seed coat dormancy and embryo dormancy can be effective^[5^]. However, on the Loess Plateau, soil water is often insufficient to support seed germination, resulting in reduced plant populations. Therefore, determining which method for breaking switchgrass seed dormancy is effective under drought stress has become a research priority for the Loess Plateau.

Switchgrass that is a polymorphic plant species with two distinct ecotypes, lowland and upland, and two main ploidy levels, tetraploid and octoploid^[8^]. Several studies have compared the practicality of methods for breaking seed dormancy of switchgrass ecotypes under benign conditions^[5,9,10^], Others have examined how germination of one particular ecotype responds to various treatments^[6,11,12^]. However, relatively few studies have examined if and how germination of switchgrass cultivars differ under varied environmental conditions, even though such information is crucial for selecting effective techniques and suitable seed of switchgrass for specific situations.

Numerous mathematical models for data analysis and prediction of seed germination responses under varied environmental conditions have been reported^[13,14^]. A number of models for calculating the time taken to reach 50% emergence (t₅₀) have been built and frequently used^[15–17^]. Recently, t₅₀ was recognized as a key parameter for calculating the theoretical base water potential for obtaining 50% germination (Y₅₀) of individual cultivars^[17^]. However, it is not know if t₅₀ estimated by modeling provides an appropriate parameter for calculating Y₅₀ for switchgrass, therefore the reliability of calculated Y₅₀ for switchgrass needs to be tested.

In this study, a comparison of two seed treatments on germination and embryo growth at optimal temperature along water potential gradients were investigated for three switchgrass cultivars including both ecotypes. The aim was to verify if and how the treatments change plant growth during the early stages of development and to examine value ofY₅₀ estimated from t₅₀.

Fig.1 Cumulative germination time courses in polyethylene glycol (PEG 6000) with different treatments at 30°C for seeds ofPanicum virgatum (switchgrass) cv. Pathfinder (a, b and c), cv. Trailblazer (d, e and f) and cv. Alamo (g, h and i). Symbols represent the observed percentages with time at each water potential. PCS, prechilling stratification; SAS, sulfuric acid scarification; CK, control.

Full size|PPT slide

2 Materials and methods

2.1 Seed treatment and experimental design

The study was conducted with seed of P. virgatum cvs Pathfinder (octoploid), Trailblazer (octoploid) and Alamo (tetraploid). The first two cultivars are classified as upland ecotype, and the last one as lowland ecotype. The seed was harvested from the Ansai Research Station of the Chinese Academy of Science. The mean 1000 seed weight for Pathfinder, Trailblazer and Alamo were 1.68±0.05, 1.85±0.07 and 1.23±0.03 g, respectively. The seed was one year old and had been stored in paper bags under cool and dry laboratory conditions. Before use, the seed was immersed in 1% sodium hypochlorite for 1 min then soaked in 75% ethanol for 3 min, and rinsed in distilled water and dried.

Prechilling stratification and sulfuric acid scarification were used to promote seed germination. Prechilling stratification was performed by refrigerating seed at 5°C for 14 days, after which the seed was washed in distilled water and air-dried. Sulfuric acid scarification was performed by soaking seed in 8 mol·L^–1 H₂SO₄ for 5 min, after which the seed was washed in distilled water and air-dried. Untreated seed was used as the control (CK).

Six water potentials (0, - 0.1, - 0.2, - 0.3, - 0.4 and - 0.5 MPa) adjusted by polyethylene glycol (PEG 6000) were used. PEG solutions were prepared by different concentrations of PEG 6000 in deionized water according to the empirical equation^[18^].

Samples of 90 seeds (three replicates of 30 seeds each) were placed on two filter papers in 90-mm Petri dishes with PEG solutions. Seeds were allowed to germinate at a constant temperature of 30°C, the temperature considered the optimal for germination of switchgrass^[19^], in a thermostatically controlled incubator (±1°C). The Petri dishes were sealed with Parafilm and placed in the dark. Germinated seed were defined as those where the root reached approximately 2 mm length. Data were collected daily until no additional germination occurred. The length of the roots and shoots of germinated seeds were measured 3 d after germination.

2.2 Measurement and verification

Total germination percentage (TGP) and mean germination time (MGT) were calculated at the end of the experiment using the following equations:

(1)$TGP\hspace{0.17em}(\%)\hspace{0.17em}=\hspace{0.17em}\left({\displaystyle \frac{n}{N}}\right)\hspace{0.17em}\times \hspace{0.17em}\mathrm{100}$

where n is the number of germinated seeds and N is the total number of seeds placed in the dish to germinate, and

(2)$MGT\hspace{0.17em}(days)\hspace{0.17em}=\hspace{0.17em}{\displaystyle \sum \frac{(n_i\hspace{0.17em}\times \hspace{0.17em}t)}{n}}$

where n_i is the number of germinated seeds at day t and n is the number of germinated seeds.

The following three equations were used to calculate the theoretical time to 50% emergence (t₅₀).

Equation (1), sigmoidal model:

(3)$y\hspace{0.17em}=\hspace{0.17em}{{\displaystyle \frac{a}{\mathrm{1}\hspace{0.17em}+\hspace{0.17em}{\left({\displaystyle \frac{x}{x_{\mathrm{0}}}}\right)}^b}}}^{ }$

where a is the peak value of y (maximum seed germination), x is days after seed germination, x₀ is days to achieve 50% of maximal seed germination, b is a calculated parameter for the curve in this sigmoidal model. Parameters were calculated by SigmaPlot 12.5 (Systat Software Inc., CA, USA). Thex value on the curve, which corresponds to 50% of cumulative seed germination (y values of the curve), was considered the theoretical time to 50% germination (t₅₀) in treatments^[20^].

Equation (2), Farooq’s model:

(4)$t_{\mathrm{50}}=t_i+{\displaystyle \frac{[(N/\mathrm{2})-n_i](t_j-t_i)}{n_j-n_i}}$

where N is the final germinated number of seed, n_i and n_j are the cumulative number of seeds germinated between adjacent counts at times t_i and t_j when n_i<N/2<n_j^[16^].

Equation (3), Mo’s model:

(5)$\mathrm{Pr}obit (GP)=a+b\lg X$

where Probit (GP) is probability unit of seed germination percentage and X is days of experiment. The a and b are estimated from observed values of Probit (GP), lgX and then t₅₀ is predicted by the equation^[15^].

2.3 Statistical analyses

A linear regression of the reciprocal of t₅₀ versus water potential was used to calculate the theoretical base water potential to reach 50% germination (Y₅₀) for each cultivar according to the value of abscissa intercept of regression line^[17^].

The data were analyzed using the statistical package SPSS 13 (IBM, Armonk, NY, USA). One-way analysis of variance (ANOVA), followed by the least significant difference test, was applied to compare treatment means. The treatment effects were considered to be significant atP<0.05.

3 Results

TGP was negatively correlated with decreasing water potential of the PEG solutions (Fig. 1; Table 1). Both prechilling stratification and sulfuric acid scarification increased TGP, and the increase with prechilling stratification on TGP was greater regardless of whether drought stress was imposed. TGP exhibited large variations among the three cultivars. Specifically, TGP of Pathfinder ranged from 88.4% to 26.7%. The highest TGP for Pathfinder corresponded to prechilling stratification without drought stress, and the lowest to- 0.5 MPa water potential stresses without seed priming. TGP of Trailblazer ranged from 71.1% to 18.9%. The highest TGP for Trailblazer corresponded to prechilling stratification without drought stress and prechilling stratification with- 0.1 MPa water potential stress, and the lowest to - 0.5 MPa water potential stresses without seed priming. TGP of Alamo ranged from 38.9% to 8.9%. The highest TGP for Alamo corresponded to prechilling stratification without drought stress, and the lowest to- 0.5 MPa water potential stresses without seed priming.

Tab.1 Effect of seed treatments and water potential (Y) on total germination percentage (n = 3), mean germination time (n = 3), and shoot (n = 10) and root (n = 10) length in germinating seeds for three cultivars of Panicum virgatum (switchgrass). P, cv. Pathfinder; T, cv. Trailblazer; A, cv. Alamo. PCS, prechilling stratification; SAS, sulfuric acid scarification

Variety	Treatment	Total germination/%	Mean germination time/d	Shoot length/mm	Root length/mm
P	Y/MPa	PCS	SAS	CK	Mean	PCS	SAS	CK	Mean	PCS	SAS	CK	Mean	PCS	SAS	CK	Mean
	0	84.4	81.1	74.4	80.0a	3.5	4.2	3.8	3.8a	22.4	19.4	19.3	20.4a	7.2	6.8	8.2	7.4a
	- 0.1	80.0	74.4	67.8	74.1ab	3.5	4.1	4.0	3.9a	21.5	18.4	18.4	19.4a	7.0	6.7	8.1	7.3a
	- 0.2	76.7	71.1	51.1	66.3ab	3.7	4.3	4.7	4.2ab	20.2	16.7	17.4	18.1a	7.0	6.0	8.0	7.0a
	- 0.3	74.4	57.8	35.6	55.9bc	3.9	4.3	3.5	3.9a	12.5	11.0	12.5	12.0b	4.3	4.8	5.1	4.7b
	- 0.4	48.9	37.8	37.8	41.5cd	3.9	4.5	4.5	4.3a	6.8	5.8	9.5	7.4c	3.5	3.8	4.7	4.0b
	- 0.5	44.4	27.8	26.7	33.0d	4.4	4.8	4.5	4.6b	4.8	5.3	7.5	5.9c	3.3	3.7	4.3	3.8b
	Mean	68.1	58.3	48.9		3.8	4.4	4.2		14.7	12.8	14.1		5.4	5.3	6.4
	Sig.	Y *			*				*				*
		Treatment ns			ns				ns				ns
		Y x treatment ns			ns				ns				ns
T	0	71.1	67.8	67.8	68.9a	3.3	3.7	3.8	3.6a	20.5	19.4	18.0	19.3a	9.5	8.3	8.3	8.7a
	- 0.1	71.1	61.1	61.1	64.4a	3.5	3.6	4.2	3.8a	20.0	18.1	17.6	18.6a	9.3	7.9	8.3	8.5a
	- 0.2	70.0	57.8	57.8	61.9ab	3.4	3.9	4.8	4.0ab	19.2	16.8	17.0	17.7a	9.2	7.8	8.1	8.4a
	- 0.3	61.1	51.1	27.8	46.7bc	3.6	4.2	4.2	4.0a	13.4	9.4	9.1	10.6b	6.4	5.7	5.8	6.0b
	- 0.4	42.2	36.7	27.8	35.5cd	4.4	4.4	5.4	4.7b	8.6	7.4	7.6	7.9c	5.7	5.2	5.7	5.5b
	- 0.5	34.4	22.2	18.9	25.2d	4.6	4.5	5.4	4.8b	6.3	5.6	5.5	5.8c	4.7	4.1	4.4	4.4c
	Mean	58.3	49.4	43.5		3.8	4.1	4.6		14.7	12.8	12.5		7.5	6.5	6.8
	Sig.	Y *			*				*				*
		Treatment ns			ns				ns				ns
		Y x treatment ns			ns				ns				ns
A	0	38.9	35.6	34.4	36.3a	4.4	4.4	4.8	4.5a	30.3	29.1	28.3	29.2a	11.6	12.3	12.9	12.3a
	- 0.1	34.4	31.1	31.1	32.2ab	4.3	4.5	4.6	4.5a	26.9	26.5	26.3	26.6b	10.7	11.3	12.2	11.4ab
	- 0.2	28.9	27.8	27.8	28.2b	4.2	4.3	4.7	4.4a	23.7	23.6	21.5	22.9c	9.9	10.5	10.4	10.3bc
	- 0.3	26.7	18.9	18.9	21.5c	4.5	4.6	4.6	4.6a	18.7	16.3	14.8	16.6d	9.5	10.1	8.1	9.2cd
	- 0.4	15.6	14.4	11.1	13.7d	4.6	4.9	5.0	4.8a	11.1	10.9	11.8	11.3e	8.9	9.2	7.9	8.7d
	- 0.5	12.2	11.1	8.9	10.7d	5.3	4.8	4.6	4.9a	9.5	9.2	9.0	9.2f	8.5	8.3	6.9	7.9d
	Mean	26.1	23.1	22.0		4.6	4.6	4.7		20.0	19.3	18.6		9.9	10.3	9.7
	Sig.	Y *			ns				*				*
		Treatment ns			ns				ns				ns
		Y x treatment ns			ns				ns				*

Note: *, Significant at P<0.05; ns, not significant.

MGT was progressively delayed by increasing drought stress (Table 1). The effects of prechilling stratification and sulfuric acid scarification on MGT weakened gradually and then disappeared with increasing drought stress. There were significant effects of drought stress on root and shoot growth. The negative effects of drought stress were increased gradually with decreasing water potential. Both prechilling stratification and sulfuric acid scarification had no obvious effect on promoting root and shoot growth regardless of whether drought stress was applied. There was large difference betweent₅₀ estimated by three equations (Table 2).

Tab.2 Time taken to reach 50% emergence (t50) for two cultivars of Panicum virgatum (switchgrass) estimated by three equations. P, cv. Pathfinder; T, cv. Trailblazer. PCS, prechilling stratification; SAS, sulfuric acid scarification

Variety	Treatment	Sigmoidal model		Farooq’s model		Mo’s model
	Y/MPa	PCS	SAS	CK		PCS	SAS	CK		PCS	SAS	CK
	0	2.99	4.18	3.62		2.60	3.88	3.28		3.78	4.31	3.58
	- 0.1	3.13	4.19	4.25		2.60	3.65	3.54		3.98	4.47	3.72
P	- 0.2	3.48	4.45	6.40		2.81	3.66	4.50		4.16	4.65	4.36
	- 0.3	3.82	5.32	–		2.83	4.12	–		4.37	5.18	–
	- 0.4	–	–	–		–	–	–		–	–	–
	- 0.5	–	–	–		–	–	–		–	–	–
	0	3.00	4.04	3.87		2.48	3.18	3.48		4.14	4.46	3.70
	- 0.1	3.33	4.30	4.76		2.71	3.09	3.57		4.25	4.64	4.15
T	- 0.2	3.50	4.60	6.12		2.73	2.98	4.35		4.22	4.99	4.31
	- 0.3	3.80	6.90	–		2.85	3.59	–		4.64	5.54	–
	- 0.4	–	–	–		–	–	–		–	–	–
	- 0.5	–	–	–		–	–	–		–	–	–

The linear regression of t₅₀ against Y was used to calculate the minimum Y for 50% germination in each treatment. For Pathfinder, thresholds of - 1.34, - 1.47 and - 0.47 MPa were estimated by the sigmoidal model for prechilling stratification treated group, sulfuric acid scarification treated group and CK, respectively,- 2.79, - 4.47 and - 0.77 MPa using Farooq’s model, and - 2.28, - 1.87 and - 1.15 MPa using Mo’s model (Fig. 2). For Trailblazer, thresholds of - 1.46, - 0.81 and - 0.54 MPa were estimated using the sigmoidal model for prechilling stratification treated group, sulfuric acid scarification treated group and CK, respectively,- 2.47, - 2.36 and - 0.85 MPa using Farooq’s model, and - 3.15, - 1.55 and - 1.33 MPa using Mo’s model (Fig. 2).

4 Discussion

This study focused on the variation in germination characteristics and early embryo growth of three cultivars of switchgrass, treated with prechilling stratification or sulfuric acid scarification and then exposed to reduced water potentials in PEG solution under laboratory conditions. A similar approach had been used to explore the effects of temperature stratification and acid scarification on seed germination inLupinus sulphureus ssp. kincaidii^[21^], Prunus serotina^[22^] and Ribes multiflorum ssp. sandalioticum^[23^]. However, relatively few reports have been published on the influence of water potential on the effects of temperature stratification and acid scarification.

Germination is known as a critical and sensitive stage in the life cycle of plants, and unsuitable environmental conditions, such as drought, greatly influence subsequent seedling establishment^[24^]. Our results showed that the TGP, and the length of root and shoot gradually declined with increasing drought intensity (Table 1). Drought restricts seed imbibition, reduces seed metabolism activation and delays seed germination^[17^], and decreased elongation of root and shoot is caused by a progressively increased gap between optimal water potential for embryo growth and ambient water potential^[25^]. Among environmental variables, soil water availability is a critical factor controlling species habitat suitability and precipitation can partially determine whether the habitat is humid enough for a specific species. Soil moisture availability is low on much of the Loess Plateau, so planting more drought tolerant cultivars of switchgrass is appropriate without suitable priming treatment and irrigation.

Fig.2 Relationships between water potential (Y) of solution and theoretical time to 50% germination (t50) in seeds of Panicum virgatum (switchgrass) cv. Pathfinder (a, b and c) and cv. Trailblazer (d, e and f) at 30°C. Data were fitted by linear regression to predict the base water potential for reaching 50% germination (Y50) under treatments.

Full size|PPT slide

The beneficial effects of seed priming on germination with increased drought stresses have been reported for various plant species, such as Chinese cabbage^[26^], sunflower^[27^] and sweet sorghum^[20^]. Dormancy imposed by seed coats can be alleviated by acid scarification^[28^], while stratification can also break seed embryo dormancy^[29^]. In the present study, prechilling stratification enhanced TGP for Pathfinder and Trailblazer under all treatments, and sulfuric acid scarification also enhanced TGP in Pathfinder and Trailblazer in most treatments (Table 1). These results indicate the existence of both embryo and seed coat dormancy in these cultivars. Similar conclusions also were drawn in previous studies with other switchgrass cultivars, Cave-in-Rock^[30^] and Kanlow^[31^]. Indeed, the promotion of TGP was better with stratification treatments in this study (Table 1). Furthermore, the effects of acid scarification treatment on TGP displayed a gradual decline with increasing osmotic stress (Table 1). Drought and the impermeable barrier imposed by seed coats can prevent root emergence due to insufficient embryo development water potential and water soluble inhibitors^[5^], resulting in extended embryo dormancy. Thus, breaking embryo dormancy is the preferred method for promoting seed germination in arid environments. In addition, Alamo had limited response to both methods tested in the present study (Table 1), which was consistent with a previous study^[32^], suggesting that, for wet sites species, some seed priming methods may not offset the germination limiting factors provided by water deficiency.

MGT can be shortened by seed priming^[27^]. Our results were consistent with previous studies, showing that seed priming methods can shorten MGT, but the effect declined or/and disappeared with increasing osmotic stress (Table 1). Furthermore, prechilling stratification and sulfuric acid scarification were not found to give improvement in root and shoot growth (Table 1). Prechilling stratification can break seed dormancy by changes to physiologic traits^[33^], while sulfuric acid scarification can dramatically alter seed coat physical properties, reducing thickness and strength thereby creating a weak area of root emergence^[34^]. Thus, prechilling stratification and sulfuric acid scarification mainly contribute to breaking seed coat or embryo dormancy, and their effect differs from hydro-priming, which has beneficial effects on MGT and root growth because of sufficient water and longer imbibition time^[27^].

Linear regression of the reciprocal of t₅₀ versus water potential was used to calculate Y₅₀ of each cultivar according to the value of abscissa intercept of the regression line^[17^]. This is a modification of the established seed analysis method. While the horizontal axis represented the temperature range of germination, the value of abscissa intercept of the regression line was used to estimate theoretical minimum temperature for germination^[13^]. Replacing temperature with water potential in the coordinate system, the value of abscissa intercept of regression line has been used to calculateY₅₀ of sorghum^[17^]. The present study showed that the range of the lowest water potential requirements to achieve 50% germination of Pathfinder and Trailblazer under prechilling stratification and sulfuric acid scarification treatments was between- 0.3 and - 0.4 MPa (Table 1). However, the linear regression estimated values of Y₅₀ were far below this range regardless of which t₅₀ was used (Fig. 2). Indeed, seeds of Pathfinder and Trailblazer proved to be difficult to germinate under the estimated Y₅₀ in subsequent attempts. Given the huge difference between predicted value and measured value, the reliability and applicable range of linear regression estimatedY₅₀ needs further investigation, specification and optimization.

5 Conclusions

The greatest benefit of seed priming was achieved by prechilling stratification, indicating that embryo dormancy may be a major limitation to switchgrass germination under drought conditions. According to the findings of our research, Pathfinder is more suitable for semi-arid environment. In addition, Alamo did not reach 50% germination with any of the treatments, showing that this cultivar is not suitable for use under drought conditions.

References

[1] Mutegi E, Stottlemyer  A L, Snow  A A, Sweeney  P M. Genetic structure of remnant populations and cultivars of switchgrass (Panicum virgatum) in the context of prairie conservation and restoration. Restoration Ecology, 2014, 22(2): 223–231

[2] Ichizen N, Takahashi  H, Nishio T ,  Liu G, Li  D, Huang J . Impacts of switchgrass (Panicum virgatum L.) planting on soil erosion in the hills of the Loess Plateau in China. Weed Biology and Management, 2005, 5(1): 31–34

[3] Sang T, Zhu  W. China’s bioenergy potential. GCB Bioenergy, 2011, 3(2): 79–90

[4] Ma Y, An  Y, Shui J ,  Sun Z. Adaptability evaluation of switchgrass (Panicum virgatum L.) cultivars on the Loess Plateau of China. Plant Science, 2011, 181(6): 638–643

[5] Duclos D V, Ray  D T, Johnson  D J, Taylor  A G. Investigating seed dormancy in switchgrass (Panicum virgatum L.): understanding the physiology and mechanisms of coat-imposed seed dormancy. Industrial Crops and Products, 2013, 45: 377–387

[6] Beckman J J, Moser  L E, Kubik  K, Waller S S . Big bluestem and switchgrass establishment as influenced by seed priming. Agronomy Journal, 1993, 85(2): 199–202

[7] Sarath G, Mitchell  R B. Aged switchgrass seed lot’s response to dormancy-breaking chemicals. Seed Technology, 2008, 30(1): 7–16

[8] Martinez-Reyna J M ,  Vogel K P . Heterosis in switchgrass: spaced plants. Crop Science, 2008, 48(4): 1312–1320

[9] Hanson J, Johnson  H. Germination of switchgrass under various temperature and pH regimes. Seed Technology, 2005, 27(2): 203–210

[10] Hacisalihoglu G. Responses of three switchgrass (Panicum virgatum L.) cultivars to seed priming and differential aging conditions. Acta Agriculturae Scandinavica Section B–Soil and Plant Science, 2008, 58(3): 280–284

[11] Ghimire S R, Charlton  N D, Craven  K D. The mycorrhizal fungus, Sebacina vermifera, enhances seed germination and biomass production in switchgrass (Panicum virgatum L). BioEnergy Research, 2009, 2(1–2): 51–58

[12] Sarath G, Hou  G, Baird L M ,  Mitchell R B . Reactive oxygen species, ABA and nitric oxide interactions on the germination of warm-season C₄-grasses. Planta, 2007, 226(3): 697–708

[13] Scott S, Jones  R, Williams W . Review of data analysis methods for seed germination. Crop Science, 1984, 24(6): 1192–1199

[14] Ritz C, Pipper  C B, Streibig  J C. Analysis of germination data from agricultural experiments. European Journal of Agronomy, 2013, 45: 1–6

[15] Mo H D. Agricultural experiment statistics. Shanghai: Shanghai Scientific and Technical Publishers, 1984 (in Chinese)

[16] Farooq M, Aziz  T, Basra S ,  Cheema M ,  Rehman H . Chilling tolerance in hybrid maize induced by seed priming with salicylic acid. Journal Agronomy & Crop Science, 2008, 194(2): 161–168

[17] Patanè C, Saita  A, Sortino O . Comparative effects of salt and water stress on seed germination and early embryo growth in two cultivars of sweet sorghum. Journal Agronomy & Crop Science, 2013, 199(1): 30–37

[18] Michel B E, Kaufmann  M R. The osmotic potential of polyethylene glycol 6000. Plant Physiology, 1973, 51(5): 914–916

[19] Hsu F, Nelson  C, Matches A . Temperature effects on germination of perennial warm-season forage grasses. Crop Science, 1985, 25(2): 215–220

[20] Patanè C, Cavallaro  V, Cosentino S L . Germination and radicle growth in unprimed and primed seeds of sweet sorghum as affected by reduced water potential in NaCl at different temperatures. Industrial Crops and Products, 2009, 30(1): 1–8

[21] Kaye T, Kuykendall  K. Effects of scarification and cold stratification on seed germination of Lupinus sulphureus ssp. kincaidii. Seed Science and Technology, 2001, 29(3): 663–668

[22] Phartyal S S, Godefroid  S, Koedam N . Seed development and germination ecophysiology of the invasive tree Prunus serotina (Rosaceae) in a temperate forest in Western Europe. Plant Ecology, 2009, 204(2): 285–294

[23] Mattana E, Pritchard  H, Porceddu M ,  Stuppy W ,  Bacchetta G . Interchangeable effects of gibberellic acid and temperature on embryo growth, seed germination and epicotyl emergence in Ribes multiflorum ssp. sandalioticum (Grossulariaceae). Plant Biology, 2012, 14(1): 77–87

[24] Muscolo A, Sidari  M, Anastasi U ,  Santonoceto C ,  Maggio A . Effect of PEG-induced drought stress on seed germination of four lentil genotypes. Journal of Plant Interactions, 2014, 9(1): 354–363

[25] Voegele A, Graeber  K, Oracz K ,  Tarkowská D ,  Jacquemoud D ,  Turečková V ,  Urbanová T ,  Strnad M ,  Leubner-Metzger G . Embryo growth, testa permeability, and endosperm weakening are major targets for the environmentally regulated inhibition of Lepidium sativum seed germination by myrigalone A. Journal of Experimental Botany, 2012, 63(14): 5337–5350

[26] Yan M. Seed priming stimulate germination and early seedling growth of Chinese cabbage under drought stress. South African Journal of Botany, 2015, 99: 88–92

[27] Kaya M D, Okçu  G, Atak M ,  Çıkılı Y, Kolsarıcı Ö. Seed treatments to overcome salt and drought stress during germination in sunflower (Helianthus annuus L.). European Journal of Agronomy, 2006, 24(4): 291–295 doi:10.1016/j.eja.2005.08.001

[28] Martín I, Guerrero  M. Effect of sulphuric acid scarification on seed accessions of cluster clover (Trifolium glomeratum) stored in a genebank. Seed Science and Technology, 2014, 42(2): 293–299

[29] Ekpong B. Effects of seed maturity, seed storage and pre-germination treatments on seed germination of cleome (Cleome gynandra L.). Scientia Horticulturae, 2009, 119(3): 236–240

[30] Shen Z X, Parrish  D J, Wolf  D D, Welbaum  G E. Stratification in switchgrass seeds is reversed and hastened by drying. Crop Science, 2001, 41(5): 1546–1551

[31] Duclos D V, Altobello  C O, Taylor  A G. Investigating seed dormancy in switchgrass (Panicum virgatum L.): Elucidating the effect of temperature regimes and plant hormones on embryo dormancy. Industrial Crops and Products, 2014, 58: 148–159

[32] Traversa A, Loffredo  E, Gattullo C E ,  Palazzo A J ,  Bashore T L ,  Senesi N . Comparative evaluation of compost humic acids and their effects on the germination of switchgrass (Panicum vigatum L.). Journal of Soils and Sediments, 2014, 14(2): 432–440

[33] Wartidiningsih N, Geneve  R, Kester S . Osmotic priming or chilling stratification improves seed germination of purple coneflower. HortScience, 1994, 29(12): 1445–1448

[34] Davis T D, George  S W, Upadhyaya  A, Parsons J . Improvement of seedling emergence of Lupinus texensis Hook. following seed scarification treatments. Journal of Environmental Horticulture, 1991, 9(1): 17–21

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31500320) and the National Science and Technology Supporting Programs (2015BAD22B01).

Compliance with ethics guidelines

Nan Wang, Jing Gao, Suiqi Zhang, and Feng Yan declare that they have no conflicts of interest or financial conflicts to disclose.

This article does not contain any studies with human or animal subjects performed by any of the authors.

RIGHTS & PERMISSIONS

The Author(s) 2017. Published by Higher Education Press. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0)