Investigating seed dormancy in switchgrass (Panicum virgatum L.): understanding the physiology and mechanisms of coat-imposed seed dormancy

Industrial Crops and Products 45 (2013) 377–387

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Investigating seed dormancy in switchgrass (Panicum virgatum L.): understanding the physiology and mechanisms of coat-imposed seed dormancy Denise V. Duclos a,∗ , Dennis T. Ray b , Daniel J. Johnson b , Alan G. Taylor a a b

Department of Horticulture, New York State Agricultural Experiment Station, Cornell University, Geneva, NY 14456-0462, USA The School of Plant Sciences, Division of Horticultural and Crop Sciences, The University of Arizona, Tucson, AZ 85721, USA

a r t i c l e

i n f o

Article history: Received 16 November 2012 Received in revised form 30 December 2012 Accepted 1 January 2013 Keywords: Embryo Germination Lemma Palea Pericarp Poaceae Seed coat Seed dormancy Switchgrass

a b s t r a c t Switchgrass (Panicum virgatum L.), a perennial warm-season grass established by seed has been recommended by the US Department of Energy as a model herbaceous biofuel crop. Neoteric seeds may be dormant resulting in delayed and sporadic germination and emergence, jeopardizing establishment of a good plant stand. Switchgrass dormancy may be alleviated by mechanical or chemical scarification, stratification, and after-ripening, suggesting physical and/or physiological dormancy. The underlying mechanisms and physiology of dormancy in these seeds; however, are not well understood. This work investigates the physiology of switchgrass seed dormancy; first by identifying the contribution of the different switchgrass structures (glumes, lemma, palea, pericarp/testa, endosperm) on regulating germination, and then by testing specific mechanisms of coat-imposed dormancy. We sequentially removed structures of freshly harvested seeds of two upland (Cave-in-Rock, Trailblazer) and two lowland (Alamo, Kanlow) cultivars. The main structure inhibiting germination was the pericarp/testa, with the lemma and palea (bracts) having a secondary effect that differed by genotype. Seeds with glumes and bracts removed, and pericarp/testa cut with endosperm either attached or removed just above the embryo, resulted in high germination percentages and rate, indicating no morphological dormancy, and no effect of the endosperm on germination. The lemma, palea, and pericarp/testa were neither a barrier for water uptake nor contained inhibitory compounds. By adjusting the oxygen concentration of the environment and the physical integrity of the covering structures, we found the enclosing structures acted as barriers to oxygen. Puncturing the pericarp/testa of seeds with glumes and bracts removed, enhanced germination at 1, 10, 21 or 100% oxygen. Combined results showed that the structural integrity of the pericarp/testa (primary) and lemma/palea (secondary) influenced germination, suggesting an important mechanical effect of these layers as barriers for radicle protrusion. Therefore, a combination of seed-coat mechanisms regulates germination in switchgrass seeds. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Switchgrass (Panicum virgatum L.) is a perennial warm-season C4 grass native to North America (it extends from Quebec, in Canada, through most of the U.S. and Central America), and is established by seed (McLaughlin et al., 1999; Parrish and Fike, 2005). Switchgrass has a large genetic diversity because of the wide range of environments in which it grows and is classified as lowland ecotypes, adapted to moist sites and warm temperatures; and upland ecotypes, adapted to dry and cooler sites Hopkins and Taliaferro (1995). Switchgrass has been used as a ground cover, forage for livestock, soil and water conservation, and wildlife habitat. The U.S. Department of Energy (DOE) recommended switchgrass as a

∗ Corresponding author at: Department of Horticulture, NYSAES Cornell University, 630W. North Street, Geneva, NY 14456, USA. Tel.: +1 607 351 8639. E-mail address: [email protected] (D.V. Duclos). 0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2013.01.005

model herbaceous biofuel crop, as switchgrass produces high yield, contains high levels of cellulose, requires low energy input for production, grows in marginal lands, and can be a dedicated biofuel crop (Moser and Vogel, 1995; Sanderson et al., 1996). Neoteric switchgrass seeds; however, like many other perennial grasses, exhibit different levels of dormancy (Blake, 1935; Robocker et al., 1953; Panciera et al., 1987; Jensen and Boe, 1991), resulting in delayed and sporadic germination and emergence, jeopardizing establishment of a good stand and limiting the use of switchgrass as a commercial crop. Seed dormancy blocks germination of an intact viable seed even under favorable conditions (Finch-Savage and Leubner-Metzger, 2006), and can be imposed by morphological, physical, and/or physiological properties of the seed (Baskin and Baskin, 2004; Finch-Savage and Leubner-Metzger, 2006). Seed dormancy can be divided into two general categories: seed coat and embryo dormancy. Seed coats can impose dormancy by interfering with water uptake and/or gas exchange, exerting mechanical restraint,

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containing chemical inhibitors, acting as a barrier against the exit of inhibitors from the embryo, or by a combination of any of these factors (Bewley and Black, 1985). Embryo dormancy can be morphological (underdeveloped) or physiological (presence of inhibitor) (Bewley and Black, 1985). In switchgrass, both treatments to break seed coat dormancy or to break embryo dormancy have proven effective. The removal or mechanical damage of the floret bracts (lemma and palea) of switchgrass manually or with abrasion using emery cloth, sandpaper, or machines (e.g., cylinder-type scarifier) (Sautter, 1962; Zhang and Maun, 1989; Jensen and Boe, 1991) has shown to improve germination in some cultivars. Exposure of switchgrass seeds to sulfuric acid (H2 SO4 : 8 M and 16.8 M) and sodium hypochlorite (NaOCl: 0.71 M) (Panciera et al., 1987; Tischler et al., 1994; Zarnstorff et al., 1994) have shown similar results. These investigations suggest that the switchgrass seed covering layers may contribute to its dormancy. Embryo dormancy is common in switchgrass, and stratification (a moist-chilling treatment) for 14 days at 5 ◦ C used before sowing increased germination in some cultivars, especially in freshly harvested seeds (Hsu et al., 1985; Zarnstorff et al., 1994; Wolf and Fiske, 1995; Shen et al., 2001). After-ripening can break dormancy, and storing several cultivars of switchgrass seeds after harvest for different lengths of time (12 weeks up to 2 years) at temperatures close to 25 ◦ C were shown to increase germination considerably when compared to neoteric seeds and seeds stored at lower temperatures or for shorter periods of time (Robocker et al., 1953; Zarnstorff et al., 1994; Shen et al., 2001). The exogenous application of the plant hormone gibberellin (Zarnstorff et al., 1994; Haynes et al., 1997; Madakadze et al., 2000; Sarath et al., 2006) and chemicals releasing reactive nitrogen species or peroxide (Sarath et al., 2006, 2007; Sarath and Mitchell, 2008) improved total germination of switchgrass seeds. The use of combined dormancy-breaking methods, like acid scarification, sodium hypochlorite, and moist-chilling, have demonstrated to have an additive effect in increasing germination (Zarnstorff et al., 1994; Haynes et al., 1997) indicating that dormancy in switchgrass is likely produced by the interaction of several mechanisms – physical and/or physiological mechanisms caused by characteristics of the seed coat and embryo. Moreover, the success of the dormancy-breaking treatments was shown to depend on the age of the seeds, after-ripening period, cultivar, temperature, cultural practices, storage conditions, seed weight, and seed size, among others (Robocker et al., 1953; Aiken and Springer, 1995; Smart and Moser, 1999; Madakadze et al., 2001; Shen et al., 2001; Hanson and Johnson, 2005). Switchgrass dormancy is a complex trait; a characteristic Simpson (1990) described in other grasses in which polymorphism relates to the nature of the seed, its environment, and changes therein. In the endeavor to understand causes and mechanisms of switchgrass dormancy, it is not sufficient for us to investigate if certain treatments break dormancy, but rather we must understand how these treatments affect dormancy. An increase in germination due to floret bract abrasion or removal, for example, does not differentiate between the different mechanisms of coat-imposed dormancy. In this work, we analyze the physiology of switchgrass seed dormancy; first by identifying the specific parts of the seed involved in restricting germination and their relative importance, and then by testing specific mechanisms of coat-imposed dormancy. This knowledge will provide important and valuable information for the improvement of dormancy-breaking treatments and for future breeding programs focused on the development of low-dormant cultivars.

2. Materials and methods 2.1. 2008–2009 Experiments 2.1.1. Seed material Mature seeds of two Panicum virgatum L. cultivars were evaluated. A dormant seed lot of the upland cultivar, Cave-in-Rock, was obtained from Ernst Conservation Seeds (Meadville, PA), and a low-dormancy seed lot of the lowland cultivar, Expresso, was developed and supplied by Prof. Brian Baldwin (Mississippi State University, Mississippi State, MS). Both seed lots were produced in 2007 and stored in paper bags at 5 ◦ C to maintain initial dormancy until experiments were carried out beginning in December of 2008. Samples of seeds from each cultivar were cleaned using a General Seed blower (Seedburo Equipment Co., Des Plaines, IL). In this study, all upland cultivars were octoploids, while the lowland cultivars were tetraploids. The word ‘seed’ will be used throughout the paper to refer to the propagule (botanical seed plus any of its covering layers: glumes, lemma, palea, and pericarp). 2.1.2. Determining seed tissues that inhibit germination To investigate the contribution of the lemma, palea, pericarp/testa, endosperm, and embryo in the dormancy of switchgrass seeds, the effect of sequentially removing these tissues was studied. Dissections were performed under a Leica MZ6 dissecting microscope by hand, using forceps, tweezers, and single sided razor blades (previously sterilized in 95% ethanol). Prior to removing the tissues, seeds were imbibed in sterile distilled water in between two layers of germination paper for 1 h at room temperature (25 ◦ C) in the dark. Seeds used were surface sterilized for 15 min on a 0.263% sodium hypochlorite solution with constant stirring and then washed with sterile distilled water three times during 5 min and placed to dry on sterile Petri dishes containing two layers of sterile germination paper overnight at room temperature before starting the experiments. Experiments included the following treatments, (1) intact caryopses (Control, Fig. 1.a), (2) glumes removed (No G, Fig. 1.b), (3) glumes and lemma removed (No G,L; Fig. 1.c), (4) glumes, lemma, and palea removed (No G,L,P; Fig. 1.d), and (5) glumes, lemma, palea, and endosperm removed (No G,L,P,E; Fig. 1.e). To investigate further the contribution of the pericarp/testa and the endosperm, experiments were performed altering the integrity of the pericarp/testa, while leaving the endosperm attached to the seed as follow, (6) seeds with glumes, lemma, and palea removed and the pericarp/testa scored around the seed in the region between the embryo and endosperm (No G,L,P SPeri; Fig. 1.f). 2.1.3. Germination test Seeds were sown in Petri dishes (100 mm × 15 mm) on two layers of sterile germination paper saturated with 3 ml sterile distilled water. Petri dishes were sealed with Parafilm® and put inside Ziploc® bags. The dishes were incubated for 7 days at a constant 30 ◦ C in the dark, and randomly placed in the germinator changing their positions daily after the measurements. The number of germinated seeds was counted daily over the 7-day incubation period. A seed was considered to have germinated when the radicle visibly protruded the seed covering layers. The number of replications and number of seeds used are specified in the figures and tables associated with each experiment. To insure that the dissection treatment did not damage the seeds, viability was assessed in seeds that did not germinate at the end of each experiment with a tetrazolium test (TZ). Seeds were cut 2/3 lengthwise using a razor blade placing the halves attached in the distal region in a 1% TZ (2,3,5-triphonyl-2H-tetrazolium chloride) solution for 12 h at room temperature (25 ◦ C). Seeds were

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Fig. 1. Seed treatments. (a) Intact caryopses, control. (b) Glumes removed, No G. (c) Glumes and lemma removed, No G,L. (d) Glumes, lemma, and palea removed, No G,L,P. (e) Glumes, lemma, palea, and endosperm removed, No G,L,P,E. (f) Glumes, lemma, and palea removed and pericarp/testa scored in the region between the embryo and endosperm all around the seed without removing the endosperm, No G,L,P SPeri. (g) Puncture of the lemma in switchgrass seeds at two locations: proximal (slightly above the edge of the embryo) or distal from the embryo. (h) Glumes removed and seed cut laterally just above the embryo to remove the endosperm, 1/2 No G. (i) Glumes removed and about a quarter of the distal part of the seed (opposite to the embryo) cut laterally, 3/4 No G. (j) Glumes, lemma, and palea removed and about a quarter of the distal part of the seed (opposite to the embryo) cut laterally, 3/4 No G,L,P. Emb: embryo.

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then assayed under the dissecting microscope following guidelines specified by AOSA (2010). At the end of the incubation time total germination percentage (TGP) and mean germination time (MGT) were calculated using the following equations, TGP (%) =

n N

× 100

(1)

where n is the number of germinated seeds at the end of the experiment and N is the total number of seeds originally placed on the plate to germinate, and MGT (days) =

 (n × t) i n

(2)

where ni is the number of germinated seeds at day t and n is the number of germinated seeds at the end of the experiment. 2.1.4. Analyzing specific mechanisms of coat imposed seed dormancy 2.1.4.1. Interference with water uptake. To investigate the effect of different seed layers on water uptake, two approaches were taken. The first experiment examined the uptake of a red tracer compound dissolved in water to compare the dormant (Cave-in-Rock) and the low-dormant (Expresso) seeds. Seeds with glumes removed (No G), and seeds with glumes, lemma, and palea removed (No G,L,P) were immersed in a 0.6% solid agarose gel with or without a tracer (Rhodamine B) (Petri dishes: 100 mm × 15 mm), and incubated at room temperature (25 ◦ C) for up to 24 h. The location and intensity of the tracer was analyzed in five seeds (per treatment, per cultivar) at 2 h, 4 h, and 24 h with a Leica EZ4D dissecting microscope equipped with a digital camera and FireCam software. Seeds were observed intact and then internally by cutting them lengthwise using a razor blade. In the second experiment, different switchgrass seed layers were examined on water uptake of dormant seeds with glumes removed (No G), seeds with glumes and lemma removed (No G,L); and seeds with glumes, lemma, and palea removed (No G,L,P). Dissection was performed after imbibing the seeds in sterile distilled water for 1 h. All the dissected seeds were dried for 24 h at room temperature (25 ◦ C) in the dark before initiating the water uptake study. Seeds were divided into five groups of 10 seeds. Each group was weighed (considered weight at time 0) and then placed on a 0.6% agar in Petri dishes (100 mm × 15 mm) and incubated at room temperature. At different time intervals the groups of seeds were blotted, re-weighted, and return to the Petri dishes for further imbibition. Measurements were conducted at 2, 4, 6, 24 and 48 h. The increase in fresh weight (IFW) (%) for each time was calculated with the following equation, IFW (%) =

(weight at timex − weight at time0 ) × 100 weight at time0

(3)

2.1.4.2. Presence of inhibitors in lemma/palea/pericarp-testa. Selected seed tissues were soaked in water or acetone or ground in water, only to extract possible chemical inhibitors. Prior to removing the selected tissues, dormant Cave-in-Rock seeds were humidified at 100% relative humidity (RH) for 24 h to avoid leakage of possible inhibitors from the seeds. The humidification was done placing the seeds on a wire mesh tray placed inside a seed box (Hoffman Manufacturing Co., Albany, OR, USA) containing water in the bottom to maintain 100% RH. Dissected seeds were separated, and 300 lemmas, 300 paleas, or 300 endosperm/pericarp/testa were placed in sterile containers containing 3.5 ml sterile distilled water or acetone, and soaked for 24 h at room temperature (25 ◦ C) in the dark. Another set was ground and soaked in water for 48 h at room temperature in the dark. One ml of the different water or acetone solutions were poured onto germination papers, cut in

half and folded in a shape of a triangle. For the acetone treatments, sterile glass Petri dishes were used and the acetone was left to evaporate at room temperature, then one ml of sterile distilled water was added to moisten the blotters. Cut embryos (Fig. 1.e) of Expresso and Cave-in-Rock were prepared and allowed to air dry for 24 h at room temperature. Dry embryos were placed on blotters treated with extracts. TGP and MGT were determined as described for Section 2.1.3. 2.1.4.3. Interference with oxygen uptake. The effect of lemma, palea, and pericarp/testa on oxygen exchange was studied using two experiments. The first experiment was performed under ambient atmospheric conditions. Seeds were dissected and/or some of them punctured, by hand, with a sewing needle. Incisions penetrated the lemma, palea, or pericarp/testa. Seeds were punctured at two locations: (1) distal – from the embryo, in the middle of the top half of the endosperm or (2) proximal – to the embryo, right above the edge between embryo and endosperm (Fig. 1.g). Five seed treatments were used: (1) glumes removed (No G), (2) glumes removed and lemma punctured (No G pL), (3) glumes removed and palea punctured (No G pP), (4) glumes, lemma, and palea removed (No G,L,P), and (5) glumes, lemma, and palea removed and pericarp punctured No G,L,P pPeri). TGP and MGT were calculated as described in Section 2.1.3. In the second experiment, only proximal punctured treatments described in the first experiment were examined in three different modified atmospheres: 1% oxygen, 10% oxygen, and 100% oxygen. Ball Elite® collection 16 oz (ca. 0.5 l) wide mouth jars were used as germination chambers. The jars were tightly closed with caps consisting of two separate pieces, a metal screw ring and a metal lid. Two one-way stopcocks with luer connections (Cole-Palmer, Vernon Hills, IL) were affixed in the lid and sealed with aquarium grade silicone to allow controlled gas exchange. The appropriate gases (99% nitrogen or 100% oxygen) were introduced into the jars through the stopcocks with both of them open; gas was introduced through one and vented by the other. Sufficient time was allowed to replace the gas volume of the chamber at least 10 times with the chosen introduced gas. Filling the chamber with 99% nitrogen, removing 60 ml of gas via syringe, and replacing it with 60 ml of 100% oxygen achieved the 10% oxygen mixture. Each chamber was tested for gas leaks prior to use and at the end of the incubation period (day 7) to assure that the gas environment remained at the indicated levels using a Pac Check bench top model 450 EC oxygen analyzer (Mocon, Minneapolis, MN). Twelve replications of 20 seeds per treatment were used, and jars were placed randomly inside the germinator. TGP was determined as previously described. 2.2. 2010 Experiments This part of the study included two dormant cultivars from both upland and lowland origins to determine if common trends were identified within genotypes and among cultivars with respect to seed layers that restrict germination. 2.2.1. Seed material Four dormant switchgrass seed lots from the 2009 production year were used: upland genotypes, ‘Cave-in-Rock’ and ‘Trailblazer’, obtained from Sharp Bros. Seed Company (Healy, KS); and lowland genotypes, ‘Kanlow’ and ‘Alamo’, obtained from Applewood Seed Company (Arvada, CO) and Ernst Conservation Seeds, respectively. Seeds were stored in paper bags at 5 ◦ C to maintain dormancy until experiments were initiated in July of 2010. A 50 g sample of seeds from each cultivar was cleaned using a South Dakota Seed blower Model D (E.L. Erickson Products, Brookings, SD).

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Fig. 2. Total germination percentage (TGP) and mean germination time (MGT) obtained after 7 days of imbibition for the dormant upland cultivar, Cave-in-Rock, (a,b) and the low-dormant lowland cultivar, Expresso, (c,d). Bars and rhomboids represent means ± SE from four replications of 20 seeds each. Statistical analyses were performed independently for each cultivar using the arcsine of TGP. Values with the same letter (in one graph) are not significantly different at the 0.05 level of probability determined by Tukey’s HSD. Black: non-viable seeds, white: dormant seeds, gray: germinated seeds.

2.2.2. Microscopy Seeds were humidified for 24 h in a 100% relative humidity chamber and then cut laterally slightly above the embryo (Fig. 1.h) with a double-edged razor blade. Dissected halves were mounted on aluminum stubs using yellow Tack-it and coated with approximately 200 nm of gold with a sputter coater. Three samples of each variety were observed with a Leica 440 scanning electron microscope. 2.2.3. Determining seed tissues that inhibit germination General methods used were described in 2008–2009 (Section 2.1.2), with additional treatments included in 2010. Intact seeds (control) were not used as glumes were not present on the Alamo and Kanlow seed lots, and in Cave-in-Rock and Trailblazer the glumes were a source of microbial contamination in germination tests. Treatments used were as follows: (1) glumes removed (No G, Fig. 1.b), (2) glumes and lemma removed (No G,L; Fig. 1.c), (3) glumes, lemma, and palea removed (No G,L,P; Fig. 1.d), (4) glumes, lemma, palea, and endosperm removed (No G,L,P,E; Fig. 1.e), (5) glumes, lemma, and palea removed and pericarp/testa scored around the seed in the region between the embryo and endosperm (No G,L,P SPeri; Fig. 1.f), (6) glumes removed and seed cut just above the embryo to remove the endosperm (1/2 No G, Fig. 1.h), (7) glumes removed and about a quarter of the distal part of the seed (opposite to the embryo) cut laterally and removed (3/4 No G, Fig. 1.i), and (8) glumes, lemma, and palea removed and about a quarter of the distal part of the seed (opposite to the embryo) cut laterally and removed (3/4 No G,L,P; Fig. 1.j). Germination tests were performed as described in Section 2.1.3. 2.3. Statistical analyses Statistical analyses were performed using SAS V. 9.1 (SAS Institute Inc., Cary, NC). All variables were tested for normal distribution and equal variances as part of the assumptions required for the

analysis of variance (ANOVA) test. When assumptions were not met, transformation of the data was performed. ANOVA was done using Proc. MIXED. Multiple comparisons of the means were done using the Tukey’s HSD (Honestly Significantly Difference) test. In the first experiment performed to determine interference with oxygen uptake, orthogonal contrasts were conducted on selected treatment comparisons. 3. Results 3.1. 2008–2009 Experiments 3.1.1. Determining seed tissues that inhibit germination To determine whether the seed structures, glumes, lemma, palea, pericarp/testa, or endosperm, influence germinability in a dormant Cave-in-Rock seed lot, different seed structures were sequentially removed and germination evaluated. Large differences were measured among treatments for total germination percentage (Fig. 2.a) and mean germination times (Fig. 2.b) (TGP: P ≤ 0.0001; MGT: P = 0.001). All treatments with intact pericarp/testa had <20% TGP (Fig. 2.a). Cutting through and removing the endosperm or altering the pericarp/testa integrity while leaving the endosperm, both had high TGPs (91% ± 1.3 and 85% ± 1.3, respectively). The smallest MGTs (fastest germination) were obtained by treatments with excised embryos and seeds with glumes, lemma, and palea removed (No G,L,P,E = 1.5 d ± 0.1, No G,L,P = 1.8 d ± 0.1), while treatments with glumes removed and intact seeds were slower (3 d ± 0.2). For the low-dormant lot of Expresso, all treatments had >90 TGP (Fig. 2.c), and treatment differences were only measured for MGT (P ≤ 0.0001, Fig. 2.d). Intact seeds had the highest MGT, 3.2 d ± 0.1, followed by seeds with their glumes removed, 1.6 d ± 0.2. All other treatments germinated faster between 1.0 and 1.2 d. Seeds with glumes and bracts (lemma + palea) were observed to have more microbial contamination than seeds in which these layers were removed.

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Table 1 Germination of Cave-in-Rock seeds, affected by different physical treatments, under normal atmospheric conditions, 21% oxygen. Puncture treatments were done either proximal or distal from the embryo (Fig. 1.g). Treatmentsa

TGP (%) Proximal

No G No G pL No G pP No G v/s No GpL and No GpPb No G,L,P No G,L,P pPeri No G,L,P v/s No G,L,P pPerib

11 ± 5 29 ± 9 35 ± 2 P = 0.002 25 ± 1 46 ± 5 P = 0.009

MGT (days) Distal 15 ± 2 13 ± 4 NS 36 ± 3 NS

Proximal 3.6 ± 0.6 3.7 ± 0.5 3.4 ± 0.4 NS 3.0 ± 0.4 3.1 ± 0.3 NS

Distal 4.4 ± 0.3 4.5 ± 0.3 NS 3.3 ± 0.2 NS

Statistical analysis was performed on total germination percentage (TGP; the arcsine of TGP used for analyses) and mean germination time (MGT) after 7 days of imbibition. Analyses were done independently for proximal and distal set of data. Values are means ± SE from four replications of 20 seeds each. a Glumes removed (No G); glumes removed and puncture lemma (No G pL); glumes removed and puncture palea (No G pP); glumes, lemma, and palea removed (No G,L,P); glumes, lemma, and palea removed, puncture pericarp (No G,L,P pPeri). b Orthogonal contrasts were performed to determine specific effects of the puncture treatment in the seeds, comparing No G with No GpL and No GpP; and No G,L,P with No G,L,P pPeri. NS: not significant at the 0.05 level of probability.

3.1.2. Analyzing specific mechanisms of coat imposed seed dormancy The experiments performed in this section intended to elucidate selected mechanisms of coat-imposed dormancy. 3.1.2.1. Interference with water uptake. Two experiments were carried out to investigate the involvement of seed layers on water uptake. In the first experiment, the two seed lots, Cave-inRock and Expresso, were imbibed in a media containing a tracer to compare layer permeability up to 24 h. No differences were observed in the pattern of the tracer uptake between the dormant and low-dormant lots with only glumes or glumes and bracts (lemma + palea) removed (data not shown). In both lots, the presence of the lemma and palea blocked the tracer diffusion to the embryo and endosperm; however, when these layers were removed, the tracer was able to penetrate the pericarp/testa into the endosperm and embryo. In the second experiment, seeds of Cave-in-Rock with glumes removed; glumes and lemma removed; and glumes, lemma, and palea removed were allowed to imbibe in a time course study for 48 h. Little differences were measured during the first 24 h among treatments, while after 48 h, no significant differences among seed treatments were observed (P = 0.288) (Fig. S1). Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.indcrop. 2013.01.005.

punctured only proximal to the embryo and TGP measured under different oxygen concentrations. Germination of the seed lot Cave-in-Rock was sensitive to oxygen concentration in the environment, as demonstrated by pooled data from the five seed treatments at the three modified atmospheres (Fig. 3) and ambient condition (Table 1). The mean TGP was 5% ± 3.3, 15% ± 3.5, 28% ± 5.9, and 21% ± 3.9 for the 1, 10, 21, and 100% oxygen environments; respectively. When statistical analysis was performed among treatments in the modified atmospheres (1%, 10%, and 100% oxygen), a significant interaction was measured between seed treatment and oxygen concentration (P = 0.0214) (Fig. 3). Multiple comparison of the TGP means (Tukey’s HSD) showed that when either lemma or palea were punctured (No G pL and No G pP) or removed (No G,L,P), germination increased significantly when compared to seeds with only glumes removed (No G) at 10% and 100% oxygen. When oxygen was limiting (1%), puncturing the lemma or palea did not increase germination compared to the non-punctured control. Puncturing the pericarp increased the TGP compared with the non-punctured pericarp treatment at each oxygen concentration. 3.2. 2010 Experiments The 2010 experiments included dormant switchgrass seed lots from two upland and two lowland cultivars to further examine the involvement of specific layers in germination/dormancy, and to

3.1.2.2. Presence of Inhibitors in lemma/palea/pericarp-testa. Experiments were performed to extract inhibitors from different layers of the seeds with water or acetone. Excised embryos were used to bioassay the presence of chemical inhibitors. Neither water nor acetone extracts were found to impair germination of low-dormant Expresso or Cave-in-Rock embryos (Table S1). Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.indcrop. 2013.01.005. 3.1.2.3. Interference with oxygen uptake. Puncturing the lemma, palea or pericarp of the domant Cave-in-Rock seeds had an effect on germination that was dependent on the proximity to the embryo (Table 1). An average 20-percentage point increase in TGP was measured after puncturing the lemma and palea proximal to the embryo, while no significant differences were measured with distal puncturing. A similar percentage point increase and trend was observed by puncturing the pericarp. MGTs were not different due to puncturing in either proximal or distal locations (Table 1). Based on these results, the second experiment was performed with seeds

Fig. 3. Total germination percentage of Cave-in-Rock seeds as affected by different physical treatments incubated for 7 days under different atmospheric conditions. All puncture treatments were done proximal to the embryo. Glumes removed (No G); glumes removed and puncture lemma (No G pL); glumes removed and puncture palea (No G pP); glumes, lemma, and palea removed (No G,L,P); glumes, lemma, and palea removed, puncture pericarp (No G,L,P pPeri). Statistical analysis performed over total germination percentage (arcsine transformed) after 7 days of imbibition. Values are means ± SE from 12 replications of 20 seeds each.

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Fig. 4. Scanning electron micrographs of laterally cut switchgrass seeds. Images show the lemma (L), palea (P), endosperm (E), and pericarp/testa, (P/T). (a) Upland dormant cultivar, Cave-in-Rock. (b) Upland dormant cultivar, Trailblazer. (c) Lowland dormant cultivar, Alamo. (d) Lowland dormant cultivar, Kanlow. (e) Low-dormant lowland cultivar, Expresso.

identify if common patterns existed among cultivars within genotypes (upland vs. lowland). 3.2.1. Microscopy The lemma and palea of upland cultivars, Cave-in-Rock (Fig. 4.a) and Trailblazer (Fig. 4.b), were as much as three times thicker

than the layers of lowland cultivars, Alamo (Fig. 4.c) and Kanlow (Fig. 4.d), at their thickest regions, where the bracts did not overlap. In upland cultivars, lemma and palea were more tightly attached to the pericarp/testa than in lowland cultivars. In all cultivars studied, the lemma and palea thickened on the basal and distal region of the seed (not shown) and in their non-overlapping

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Fig. 5. Total germination percentage (TGP) and mean germination time (MGT) obtained after 7 days of imbibition for four different switchgrass cultivars in which seeds were subjected to different physical treatments. Cultivars: upland, Cave-in-Rock (a,b) and Trailblazer (c,d); lowland, Alamo (e,f) and Kanlow (g,h). Bars and rhomboids represent means ± SE from eight replications of 20 seeds each. Statistical analyses were performed independently for each cultivar using the arcsine of TGP. Values with the same letter (in the same graph) are not significantly different at the 0.05 level of probability determined by Tukey’s HSD. Black: non-viable seeds, white: dormant seeds, gray: germinated seeds.

areas, while the pericarp/testa remained relatively constant and was highly attached to the endosperm and embryo. In all cultivars, the lemma is slightly thicker than the palea. The thickness of the lemma and palea of dormant, lowland cultivars, Alamo and Kanlow, were similar to those of the low-dormant, lowland cultivar, Expresso, (Fig. 4.e). 3.2.2. Determining seed tissues that inhibit germination Four seed lots representing two upland and two lowland cultivars showed strong statistical evidence for differences among treatments (each seed lot was analyzed independently, P ≤ 0.0001) (Fig. 5.a,c,e,g). The consistently highest TGP for all seed lots were obtained when glumes, lemma, palea, and endosperm were removed or when glumes, lemma, and palea were removed and the pericarp/testa scored. The TGP mean for these two treatments were 66% for Cave-in-Rock, 93% for Trailblazer, 80% for Alamo, and 85% for Kanlow. For comparison within the same cultivar, the 2010 Cave-in-Rock seed lot had 64% ± 3.8, while the 2007 Cave-in-Rock

seed lot had 91% ± 1.3 for the No G,L,P,E treatment. TZ tests showed that the majority of the embryos that did not germinate were dormant in the 2010 seed lot. In both upland seed lots, germination was not affected when the lemma or lemma and palea were removed, compared to seeds with only glumes removed (Fig. 5.a,c). In contrast, TGP increased by about 30 percentage points in both lowland seed lots for the same treatment comparison (Fig. 5.e,g). In Trailblazer (Fig. 5.c), germination increased about 20 or 50 percentage points from seeds with lemma and palea that were cut laterally removing either ¼ (3/4 No G) or ½ (1/2 No G) of the seed, respectively, compared to seeds with only glumes removed. Germination, however, was not affected for the same treatment comparison in Cave-in-Rock (Fig. 5.a). For lowland seed lots, TGP for 3/4 No G and 1/2 No G seeds increased by 40 and 50 percentage points, respectively for Kanlow (Fig. 5.g); and by 20 and 40 percentage points, respectively for Alamo (Fig. 5.e), when compared to No G seeds. Three treatments had glumes, lemma, and palea (G,L,P) removed: only G,L,P

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removed (No G,L,P); G,L,P removed and the distal ¼ removed (3/4 No G,L,P); and G,L,P removed and the pericarp/testa scored to break integrity (No G,L,P SPeri). In both upland seed lots, the TGP was lowest in the control (No G,L,P), increased with removal of the distal ¼ of the seed, and was highest with scoring the pericarp/testa (Fig. 5.a,c). In both lowland seed lots, the control TGP was lower than the other two treatments. Mean germination times were significantly different among treatments within each seed lot (P ≤ 0.0001) (Fig. 5.b,d,f,h). The slowest treatment for all seed lots was seeds with only glumes removed (MGT = ∼3 d). Treatments with the lemma and palea still attached also had slow germination rate. Across all treatments, the upland seed lots had slightly slower germination rates (Cave-in-Rock = 2.4 d, Trailblazer = 2.3 d) than lowland seed lots (Kanlow = 1.9 d, Alamo = 2.2 d).

4. Discussion and conclusions 4.1. Identification and relative importance of seed structures involved in restricting germination Species in the Poaceae family, posses a non-deep physiological dormancy, according to the description of the different types of dormancy and their classification (Baskin and Baskin, 2004; Finch-Savage and Leubner-Metzger, 2006). As reviewed in the introduction, switchgrass dormancy could be imposed by the interaction of the embryo and its seed covering layers; however, which enclosing structures of the embryo and to what extent germination was affected has not been investigated. In this study, a series of experiments were undertaken to elucidate which layers of the seed are involved in switchgrass seed dormancy, their relative importance, and their proposed mechanisms. In all dormant seed lots examined and under environmental conditions that induced dormancy (30 ◦ C constant), embryos excised almost fully from their covering layers exhibited between 70% to over 90% germination, indicating that the seed tissues enclosing the embryo play a primary role in regulating germination and dormancy (Fig. 2, Fig. 5). Furthermore, by sequentially removing the enclosing seed structures, it was determined that the integrity of pericarp/testa played the major role in all dormant seed lots, while the lemma and palea played a secondary role that differed among upland and lowland seed lots. The effect of these layers on dormancy were reported to affect germination in other grasses including Tripsacum dactyloides L. (Tian et al., 2002), Buchloe dactyloides (Ahring and Todd, 1977), Andropogon scoparius and A. gerardii, Sorghastrum nutans L., Bromus inermis (Coukos, 1944), Chloris cucullata and C. subdolichostachya (Herrera-Cedano et al., 2006). Even though in most seed lots dormancy was predominantly coat-imposed, intrinsic factors within the embryo were still present, as a small percentage were unable to germinate even when enclosing structures were removed (Cave-in-Rock from 2010 experiment). In upland seed lots of Cave-in-Rock and Trailblazer, completely removing the lemma and palea alone did not significantly increase germination (Fig. 5.a,c). Removing the lemma and palea completely or removing small parts of them increased germination only when the pericarp/testa integrity was altered. The same results were observed by Probert et al. (1985) in Dactylis glomerata. These findings indicate a synergistic secondary role for the lemma and palea with respect to the pericarp/testa on germination. Altering the pericarp/testa integrity increased germination depending on the proximity to the embryo. In treatments with bracts still attached, 1/2 No G seeds had higher TGP means that 3/4 No G seeds; and in treatments with glumes and bracts removed, No G,L,P SPeri seeds had better germination than 3/4 No G,L,P seeds. It was not possible

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to conclude if this effect was due to the actual location of the alteration or to the extent of it; however, because different responses in germination were also observed when the pericarp/testa was punctured either in a proximal or distal position from the embryo (Table 1); results suggest that the effect is related to the location of the alteration. In dormant lowland cultivars, Alamo and Kanlow, removing the lemma and palea alone increased germination by about 30 percentage points, demonstrating an independent effect of these layers on germination (Fig. 5.e,g). However, as in upland cultivars, removing a small section of the lemma and palea was equally or more effective than its complete removal, as it was accompanied with cutting of the pericarp/testa; supporting also a synergistic effect. Altering the pericarp/testa increased germination independently of the proximity of their damage to the embryo. Simpson (1990), reviewed the effect of lemma and palea on 25 different grasses species with seed dormancy, and found that removing these layers only increased germination to 100% in only one out of 54 comparisons, while in the remainder, their removal improved germination by about 25%, similar to the results obtained in lowland cultivars of switchgrass in our study. The endosperm did not affect dormancy, as scoring through the pericarp (No G,L,P SPeri) had a similar germination as removing the endosperm (No G,L,P,E) (Fig. 2 and Fig. 5). Germination of half seeds (1/2 No G), in which almost all the endosperm was removed (the same amount as in excised embryos) was either higher or equal to seeds with only a small portion of the endosperm removed (3/4 No G). Hence; it is the integrity of the pericarp/testa and not the removal of the endosperm that impacted germination. The earlier literature is misleading on the effect of endosperm removal on dormancy as illustrated on Leymus chinensis (Ma et al., 2008) and Leymus racemosus (Huang et al., 2004). Both studies concluded that the endosperm was a critical factor affecting dormancy as germination increased as the proportion of endosperm was removed. However, neither study distinguished between damaging the pericarp/testa, while leaving the endosperm, and damaging the pericarp/testa that accompanied removing the endosperm. In the low-dormant cultivar, Expresso, TGP means for all seed treatments were above 90%. The presence of the covering structures, lemma, palea and pericarp/testa did not interfere with germination as it did in the dormant cultivars studied. The SEM images revealed that the bracts (lemma and palea) and pericarp/testa in Expresso seeds were similar in their thickness to the lowland cultivars, Alamo and Kanlow (Fig. 4). Therefore, the low-domancy trait of Expresso may reside in the embryo. Overall, seeds with glumes and with bracts (lemma and palea), even in the low-dormant cultivar, Expresso, had a slower germination rate than seeds without these covering structures or excised embryos. This is important when considering establishment in the field as rapid germination improves switchgrass competition with annual grassy weeds (Zarnstorff et al., 1994). 4.2. Identifying specific mechanism of coat-imposed dormancy Increased germination was measured in all dormant seed lots tested when the enclosing seed structures were removed, cut, or punctured. These findings and previous studies support coat-imposed dormancy regulating germination in switchgrass (Sautter, 1962; Panciera et al., 1987; Zhang and Maun, 1989; Jensen and Boe, 1991; Tischler et al., 1994, Zarnstorff et al., 1994). Seed covering layers may act as a barrier for water uptake, leakage of inhibitors, gas exchange, or radicle protrusion, as well as a source of inhibitors (Bewley and Black, 1985). Interference of water uptake is common in seeds of different families (e.g., Fabaceae, Cannaceae, Convolvulaceae, Chenopodiaceae, and Malvaceae) with hard seed coats. In most cases, the testa was

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primarily responsible of restricting the water uptake due to its waxy cuticles, suberin, thick-walled palisade and osteosclereid layers (Bewley and Black, 1985). Conversely, switchgrass seeds had a soft and thin pericarp/testa, but strong lemma and palea (Fig. 4). In our study, the lemma and palea slightly decreased water uptake during the first 36 h (Fig. S1). A similar result was reported for Panicum turgidum (Koller and Roth, 1963). However, after 48 h of imbibition the moisture content of seeds with no glumes was not different from seeds with lemma and palea removed, thus providing further support that water moves freely through the seed covering layers and does not interfere with water uptake, as suggested by Jensen and Boe (1991). Related to water uptake is seed covering permeability to solute diffusion during imbibition. Based on Salanenka and Taylor (2009), the seed coat of switchgrass is semi-permeable; similar to tomato, pepper, and onion; characterized by permeability to moderately, lipophilic compounds that are non-ionic in nature and impermeable to ionic or zwitterionic compounds, like Rhodamine B; the tracer used to compare water movement between dormant and low-dormant seeds. Our study was able to differentiate which layers in switchgrass seeds were responsible for the semi-permeable characteristic. The bracts (lemma and palea), and not the pericarp/testa restricted tracer movement through the embryo and endosperm. Moreover, this semi-permeable characteristic was present in both dormant (Cave-in-Rock) and low-dormant (Expresso) seed lots. Chemical inhibitors are generally absent or have minimal effect in the Poaceae family. Simpson (1990) summarized results from nine investigations, and chemical inhibitors were not found in five studies, while the other four studies produced ambiguous evidence for the presence of inhibitors. For example, in Dactylus glomerata, inhibitors contained in the lemma, palea and caryopses inhibited lettuce seedling growth, but extracts were not examined on D. glomerata seeds (Fendall and Canode, 1971). Probert et al. (1985) examined lemma and palea extracts and concluded that it seemed unlikely that dormancy could be related to the inhibitor content of these tissues in D. glomerata. The strongest evidence for water-soluble compounds that inhibited germination was reported from the husks of Triticum aestivum (Kato et al., 2002) and Triticum tauschii (Gatford et al., 2002); and from the endosperm of Uniola paniculata (Westra and Loomis, 1966). Nine extracts were examined in our study and none of them reduced germination by over 10% (Table S1). Moreover, the removal of small parts of the lemma and palea was equally or more effective than their complete removal in promoting germination (Fig. 5), as also reported by Probert et al. (1985) in D. glomerata. These collective results indicated that the seed coat layers were not a source of inhibitors. Many studies have suggested that coat-imposed dormancy in some grasses was the result of a lack of oxygen diffusion to the embryo (Vose, 1956; Roberts, 1964; Mott and Tynan, 1974; Renard and Capelle, 1976). In some of these studies, conclusions were made based on the positive effect of oxidizing agents (e.g., hydrogen peroxide), and the measurement of oxygen uptake by intact or de-hulled seeds during germination (Aristida contorta, Mott and Tynan, 1974; Brachiaria ruziziensis, Renard and Capelle, 1976). However, as noted by Simpson (1990), even though in many cases oxygen is involved in dormancy breaking, results are not conclusive, as experiments did not properly test whether increased oxygen actually broke embryo dormancy. In our study, changing both the oxygen concentration and the physical integrity of the covering structures of the embryo, tested the effect of seed coat as a barrier for oxygen uptake or availability. Increasing the oxygen level from 1, to 10 to 21% increased mean germination of all treatments (Table 1, Fig. 3), indicating that the embryo itself is responsive to oxygen concentration. However, germination at 100% oxygen was not greater than under ambient conditions. The

stimulatory effects of oxygen on germination were found in other species including Aristidia contarta (Mott and Tynan, 1974), Dactylis glomerata (Probert et al., 1985), Setaria faberii (Dekker and Hargrove, 2002), and Hordeum vulgare (Benech-Arnold et al., 2006). However, in all these cases, either intact seeds (first three studies) or embryos (last study) were studied, so the effect of seed structures and oxygen could not be examined. The particular mechanism by which oxygen availability affects germination in switchgrass seeds is still unclear; however, it seems that it is related to biochemical pathways involved with oxidative reactions, since hydrogen peroxide and oxidative agents were shown to increase germination in some dormant cultivars (Sarath et al., 2006, 2007; Sarath and Mitchell, 2008). The molecular mechanisms by which these exogenous agents induce germination are under investigation (Diaz and Isabel, 2011). Puncturing the lemma, palea, and pericarp/testa proximal but not distal to the embryo increased the percent germination under ambient conditions (Table 1). The increase in germination was attributed to enhanced gas exchange adjacent to the embryo. However, germination was much greater when removing the endosperm or scoring the pericarp compared to the punctured treatments (compare Table 1 with Fig. 2). This indicates an important mechanical characteristic of the pericarp/testa on regulation of germination. Therefore, the pericarp/testa appears to act as a barrier for radicle emergence. 4.3. Conclusions The combined results of this study demonstrated that switchgrass seed dormancy and germination were highly variable among different genetic backgrounds, similar to other grasses, including Panicum coloratum L. (Tischler and Young, 1987) and in other studies on switchgrass (Robocker et al., 1953; Aiken and Springer, 1995; Smart and Moser, 1999; Madakadze et al., 2001; Hanson and Johnson, 2005). However, important overall conclusions could be drawn from this research. In both upland and lowland cultivars studies, seed coat-imposed dormancy was the main factor contributing to lack of germination in the non-deep dormant switchgrass seeds; however, the presence of physiological embryo dormancy and its interaction with its surrounding covering structures, can not be discarded (also the case in other grasses like Zoysia japonica Steud., Li et al., 2010). Studies involving the same cultivars and the effect of hormonal, temperature, and light factors on the germination of intact and physically altered seeds were evaluated, and are presented in another paper. With regards to seed coat-imposed dormancy, the main structure inhibiting germination was the pericarp/testa, while the lemma and palea had a secondary effect that behaved differently in upland and lowland cultivars. The effect of the endosperm in restricting germination was absent in all cultivars tested. In the identification of specific mechanisms of coat-imposed dormancy performed with dormant seeds of the upland cultivar, Cave-in-Rock, covering layers were not barriers for imbibition as the lemma and palea only slightly retarded water uptake during the initial 36 h. The seed covering layers were not a source of inhibitors; however, the lemma and palea could restrict the leakage of inhibitors from deeper tissues of the caryopses. Evidence supported a physical barrier to oxygen and most likely to radicle protrusion. Collectively, dormancy is the culmination of several morphological structures and mechanisms. Acknowledgements We would like to thank Dr. Brian Baldwin, Ernst Conservation Seeds, Sharp Bros. Seed Company, and Applewood Seed Company

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