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SEED PHYSIOLOGY, PRODUCTION & TECHNOLOGY

Rapid and Effective Germination Methods for Overcoming Seed Dormancy in Annual Canarygrass

Dep. of Plant Sciences and Crop Development Centre, Univ. of Saskatchewan, 51 Campus Dr., Saskatoon, SK, Canada S7N 5A8

^* Corresponding author (hucl{at}sask.usask.ca)

	ABSTRACT

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Dormancy in pedigreed Phalaris seed can lead to unsatisfactory germination in seed testing. The objective was to determine the effectiveness of recommended germination methods in overcoming seed dormancy in annual canarygrass (P. canariensis L.). In 2003, ‘CDC Maria’ was grown at two locations in Saskatchewan, Canada. At maturity, panicles were harvested and stored at –20°C. Four replications of 50 seeds were used in each experiment. Hulled and hand dehulled seed were (i) stored at 23°C for 0 to 16 wk before germinating at 23°C, (ii) germinated at 15 to 30°C (with and without light), (iii) allowed to imbibe water at 23°C for up to 96 h, (iv) treated with pricking, hydrogen peroxide (H₂O₂), gibberellic acid (GA), ethephon (2-chloroethylphosphonic acid), potassium nitrate (KNO₃), chilling, and heating before germinating at 23°C, and (v) treated with KNO₃, heating, and chilling before germinating (with and without light) at 15°C, 15/25°C (16/8h), and 20/30°C (16/8h). Mean germination was tested to be significantly different (P = 0.05) from 95% germination by t tests. The after-ripening period required to overcome primary dormancy in hulled seed was 8 to 10 wk longer than in dehulled seed. Hulled seed developed deeper secondary dormancy compared with dehulled seed, particularly at 20 and 25°C, with neither displaying <95% germination at 15°C or >10% at 30°C. Dehulled seed imbibed sufficient water to initiate germination 2 d faster than hulled seed. Applications of H₂O₂ and GA overcame dormancy in dehulled seed, while none of the treatments overcame hulled seed dormancy. Dehulled and hulled seed displayed unsatisfactory germination, regardless of treatment or light regime, when germinated under an alternating temperature regime of 20/30°C. We recommend (i) hand dehulling seed, (ii) treating hulled seed with 0.01M KNO₃, or (iii) treating hulled seed with heat (3 d at 30°C) before germinating at 15°C for 10 d in darkness on top of blotters to overcome seed dormancy rapidly and effectively in annual canarygrass.

Abbreviations: AOSA, Association of Official Seed Analysts • BB, between blotters • CFIA, Canadian Food Inspection Agency • GA, Gibberellic acid • ISTA, International Seed Testing Association • KER, Kernen Crop Research Farm • SF, Seed Research Farm • TB, top of blotters

	INTRODUCTION

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IN PHALARIS, seed dormancy has been reported in five annual (Phalaris canariensis, Matus-Cádiz et al., 2001; P. brachystachys Link., Jimenez-Hidalgo et al., 1995; P. paradoxa L., Drennan and Bain, 1987; P. minor Retz., Singh and Dhawan, 1976; P. aquatica L., Young et al., 1973) and one perennial species (P. arundinacea L., Junttilla and Nilsen, 1980). Annual canarygrass (P. canariensis), grown for feeding caged birds, is the only annual Phalaris species that has gained commercial importance (Putnam et al., 1996). Phalaris brachystachys, a direct ancestor of annual canarygrass (Carlson et al., 1996), P. paradoxa, and P. minor occur as weeds in cultivated crops, while the other two Phalaris spp. are used as forages in cool temperate (P. arundinacea) and Mediterranean climates (P. aquatica) (Carlson et al., 1996).

Seed dormancy, the temporary failure of a viable seed to germinate (Simpson, 1990), reduces germination percentages in seed testing of annual canarygrass (Phalaris canariensis; Matus-Cádiz et al., 2001). Satisfactory germination percentages are difficult to obtain from freshly harvested samples, particularly in years when grain filling occurs under cool growing conditions. Pedigreed seed must be guaranteed for a high level of germination and, thus, determining which germination method, if any, promotes the highest germination percentages in dormant seed, during a short test period, is of interest. Dormancy can be overcome in P. canariensis (Matus-Cádiz et al., 2001), but no evidence to date indicates which seed test most effectively overcomes dormancy. Standard germination methods recommend two [Canadian Food Inspection Agency (CFIA), 1997; International Seed Testing Association (ISTA), 1985] or no treatments [Association of Official Seed Analysts (AOSA), 2002] before exposing seed to an alternating temperature regime using between blotters (BB) or top of blotters (TB) substrata (Table 1).

	MATERIALS AND METHODS

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Annual canarygrass cv. CDC Maria, possessing glabrous hulls and brown caryopses (Matus-Cádiz et al., 2003), was obtained from the Crop Development Center (Univ. of Saskatchewan, Saskatoon SK). In 2003, CDC Maria was grown at the Seed Research Farm (SF) and Kernen Crop Research Farm (KER, Univ. of Saskatchewan) in 1.2 x 7.5 m strips. Four strips were seeded on 14 May (SF) and 21 May (KER) at a rate of 250 seeds m^–2 on fallow land. Fertilizer (11-51-0, N-P-K) was drilled in with the seed at a rate of 50 kg ha^–1. The soil type was a Dark Brown Chernozem clay loam at the SF and a Dark Brown Chernozem (Typic boroll) clay, clay loam at KER. At Zadoks growth stage 92 (Zadoks et al., 1974), 1000 panicles were harvested from the upper canopies of each strip, air-dried in darkness at 23°C (40% relative humidity) for 7 d, and then stored at –20°C until needed. In contrast, nondormant control seed lots from both locations were obtained by storing intact panicles at 23°C for 18 wk. Germination tests were conducted within 32 wk of harvest.

Germination tests followed standard protocols (CFIA, 1997), unless otherwise specified. Panicles from each strip were hand-threshed, unless otherwise specified, to avoid split hulls and damaged seed. Cracked, shriveled, and immature seed were discarded. The hull (palea and lemma) enclosing the seed was retained in samples denoted hulled seed. Hulls were removed with (i) gentle rubbing between two layers of smooth rubber sheeting (denoted hand dehulled seed) or (ii) a ribbed rubber-belted de-awner (denoted mechanically dehulled seed). Samples denoted hand dehulled (with hulls) had their threshed hulls placed onto the substrata at germination time to test for the possible presence of inhibitors.

Seeds were placed in 60- x 15-mm Petri dishes each containing one (TB) or two Whatman No. 1 filter papers (BB). For TB substrata, seeds were placed on top of the filter paper, while seeds were placed between two filter papers for BB substrata. Filter papers were moistened with distilled water (2 mL), unless otherwise specified. Distilled water was used for subsequent moistening of filter papers for all treatments tested. Four replications of 50 seeds per treatment were used in each experiment. Seeds were uniformly spaced on the filters so that contact between adjacent seeds was avoided and Petri dishes were placed in a large plastic container containing water-saturated towels before placing into incubators (Hotpack Corp., Philadelphia, PA; model 352632). Seeds were considered germinated once the radicle had emerged by 2 mm from the seed coat. Percentage germination was defined as (number of germinated seeds/50 imbibed seeds) x 100. For each location, percentage data were averaged over four replications and the means were tested to be significantly different (P = 0.05) from each other by t tests (Minitab Inc., 2000). The six laboratory experiments conducted are described below.

Experiment 1: After-Ripening of Mechanically Dehulled, Hand Dehulled, and Hulled Seed
Hulled seed was removed from storage at –20°C, dehulled (if needed), and then stored at 23°C (40% relative humidity) for 0 to 16 wk to determine the after-ripening period required to overcome dormancy. Following storage at 23°C, seeds were simultaneously imbibed with distilled water and incubated in darkness at 23°C. Final counts were taken at 14 d.

Experiment 2: Effect of Temperature on Overcoming Dormancy in Hand Dehulled and Hulled Seed
Hulled seed was removed from storage at –20°C, dehulled (if needed), imbibed with distilled water, and then incubated at 15, 20, 25, or 30°C under a photoperiod with (16/8 h, dark/light) or without light. Only TB substrata were tested. Light was provided by two 115 W cool-white florescent sources vertically oriented on opposite sides of the germinator. A photosynthetically active radiation level of 17 µmol m^–2s^–1 reached the surface of Petri dishes within plastic containers. Final counts were taken at 14 d.

Experiment 3: Effect of Substrata Types on Germination of Hand Dehulled and Hulled Seed
Two substrata types (TB and BB) were assessed for their ability to promote germination in hand dehulled (without and with hulls) and hulled seed. Hulled seed was removed from storage at –20°C, dehulled (if needed), imbibed with distilled water, and then incubated in darkness at 23°C. Water uptake rate of dehulled (without and with hulls) and hulled seed was measured after 24, 48, 72, and 96 h at 23°C in darkness. Four replications of 50 seeds were used per treatment. Seeds were blotted to remove excess surface solution and weighed immediately at each sampling time. Care was taken not to remove too much treatment solution as the seeds were returned to their appropriate dish after each sampling time. Water uptake (%) was defined as [(wet weight – initial air dry weight)/wet weight] x 100. Differences in mean water uptake between dormant vs. nondormant seed, within a treatment, were tested to be significantly different at P = 0.05 using paired t tests. Water uptake determinations were terminated after 48 h for dehulled seed samples and 96 h for hulled seed samples once germination was 5% (P = 0.05) based on one-tailed t tests. Final counts were taken at 10 d, instead of 14 d, in Exp. 3 to 6 as our objective was to develop a rapid method (14 d).

Experiment 4: Effect of Treatments on Water Uptake in Hand Dehulled Seed
Hydrogen peroxide (BDH Inc., Toronto, ON; Cat. No. ACS 399), GA (Sigma-Aldrich Corp., St. Loius, MO; Cat. No. G-7645 with at least 90% GA₃), and ethephon (Bayer CropScience, Regina, SK; ETHREL brand with 240 g L^–1 of active ingredient) were tested to determine their effects on water uptake (24 and 48 h) and in promoting germination of hand dehulled seed. Hydrogen peroxide, an oxidizing agent, promotes germination in reed canarygrass (P. arundinacea L.; Junttilla and Nilsen, 1980), while GA is frequently recommended for promoting seed germination in many grasses (ISTA, 1985; CFIA, 1997; AOSA, 2002). The use of ethephon is rarely recommended for promoting germination (CFIA, 1997; AOSA, 2002). The six treatments tested, using only TB substrata, were as follow: 0.44 M H₂O₂; 0.88 M H₂O₂; 0.72 mM GA; 1.44 mM GA; 0.2 mM ethephon, and 1 mM ethephon. Higher concentrations of H₂O₂ were employed, but resulted in seed damage (data not shown). All treatments were dissolved in distilled water, except 1.44 mM GA treatments dissolved in 0.01 M sodium phosphate (CFIA, 1997). Ethephon treatments followed the methods described by the AOSA (2002). Hulled seed was removed from storage at –20°C, dehulled (if needed), and then incubated in darkness at 23°C following treatments. Water uptake (%) at 24 and 48 h was collected as described above. Final counts were taken at 10 d.

Experiment 5: Effect of Treatments on Water Uptake Time in Hulled Seed
The three exogenous chemicals described in Exp. 4 were tested along with KNO₃ (Sigma Cat No. P-6083) and 2.33 mM GA to determine if they promoted water uptake (24, 48, 72, and 96 h) and germination in hulled seed. Potassium nitrate is frequently recommended for promoting germination in dormant seed (ISTA, 1985; CFIA, 1997; AOSA, 2002). The 0.01 M KNO₃ treatment followed the methods described by the CFIA (1997). Hulled seed was removed from storage at –20°C, imbibed with treatment solution, and then incubated in darkness at 23°C. Water uptake (%) at 24, 48, 72, and 96 h was determined as described above. Final counts were taken at 10 d.

The mechanical treatment of pricking the caryopsis hull once (one side of caryopsis) or twice (opposite sides of caryopsis) with a pin was tested to determine if pricking promoted water uptake over time (24, 48, 72, and 96 h) and germination of hulled seed. Seeds were imbibed with distilled water and incubated in darkness at 23°C for 24 h. After 24 h, the hulls were pricked at the caryopsis midpoint just enough to puncture the hull and outer seed layer. A stereoscope was used during pricking to avoid damaging the embryo. The lemma was thicker than the palea. Water uptake (%) at 24, 48, 72, and 96 h was determined as described above. Final counts were taken at 10 d.

Experiment 6: Effect of Temperature, Light, Substrata, and Treatments on Germination of Hand Dehulled and Hulled Seed
Various temperature regimes (15°C; 15/25°C; 20/30°C), light regimes (24 h darkness and 16/8h, dark/light), substrata (TB; BB), and dormancy breaking treatments (0.01 M KNO₃; chilling; heating) were assessed for their effectiveness in overcoming dormancy in hand dehulled (without and with hulls) and hulled seed. Chilling (ISTA, 1985; CFIA, 1997; AOSA, 2002) and heating (ISTA, 1985; CFIA, 1997) are frequently recommended for promoting germination in dormant seed. For chilling, seeds were imbibed in distilled water at 23°C for 24 h (in darkness) before transferring to 5°C for 3 d (in darkness). For heating, seeds were heated at 30°C with an Isotemp oven (Fisher Scientific Ltd., Ottawa, ON; Model 655G; air was circulated with a portable fan), for 3 d before imbibing seeds using distilled water. Average moisture content of dehulled grain samples was 11.5% (SE ± 0.2) before and 7.0% ± 0.3 after heating. Chilling or heating periods were not included in the 10-d germination period. Following treatments, seed was imbibed with water, unless otherwise specified, and incubated at one of three temperature regimes (with or without light) using TB or BB. Light was applied during the high temperature period for 8 h each day as described for Exp. 2. Counts were taken at 2-d intervals over a 10-d period, with final counts taken at 10 d.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
After-Ripening of Mechanically Dehulled, Hand Dehulled, and Hulled Seed
The after-ripening period refers to the number of weeks of storage at 23°C it takes seed to lose its dormant state following harvest (Simpson, 1990). Dehulled and hulled seed from KER required a longer period to after-ripen, compared with SF seed, indicating that seed from KER possessed deeper primary dormancy levels (Fig. 1). Primary dormancy refers to the dormancy initiated in early seed development that persists for different lengths of time depending on genotype and environment (Simpson, 1990). In 2003, average temperature during grain-filling (July) and seed maturation (August) ranged from 18°C (July) to 20°C (August) at KER and from 20°C (July) to 22°C (August) at the SF. Generally, low temperatures during grain-filling and seed maturation in grasses result in increased levels of primary seed dormancy in the mature seed (Simpson, 1990). Singh et al. (1999) reported that dormancy in Phalaris species require 24 to 52 wk of after-ripening to overcome their dormant state, suggesting that primary dormancy levels in annual canarygrass may be shallower than in its related species.

View larger version (23K): [in this window] [in a new window]   Fig. 1. Effect of duration of storage, before germination at 23°C for 14 d, on germination percentage in dormant, hulled, hand-dehulled, and mechanically dehulled seed grown at Kernen (KER) and Seed Farm (SF) in 2003. Bars represent standard errors to compare treatments within each sampling day. For non-dormant seed lots from KER and SF, germination percentages were 95% (P = 0.05) at 0 wk (data not shown).

In grasses, most increases in germination following damage of the seed coat are attributed to increased permeability to O₂ and, thus, availability of O₂ to the embryo (Simpson, 1990). Mechanically dehulled seed lost its primary dormancy 2 wk (SF) to 4 wk (KER) faster than hand dehulled seed (Fig. 1). This differential loss of dormancy likely results from pericarp scarification (Simpson, 1990), suggesting that the pericarp may play a role in the observed seed dormancy. Mechanically dehulled seed displayed reduced germination (about 90%) likely because mechanical dehulling resulted in embryo damage. Similarly, Vose (1956) reported that seed scarification with sand paper improved germination to 50% in a P. aquatica x P. arundinacea hybrid when tested at 25°C (with light) for 21 d.

Effect of Temperature on Overcoming Dormancy in Hand Dehulled and Hulled Seed
Deeper dormancy in grasses is expressed as a broadening of the range of temperatures, either side of some optimal value, in which germination is inhibited (Simpson, 1990). With and without light, seeds exhibited deep dormancy at temperatures higher than 15°C (Fig. 2), suggesting that 15°C is optimal for germinating P. canariensis. Similarly, Drennan and Bain (1987) reported 15°C as an optimum for germinating dormant caryopses of P. paradoxa. Germination percentages in dehulled samples were reduced in the presence of light at 20 and 25°C (Fig. 2), confirming that light can inhibit germination in Phalaris (Nakamura, 1962; Landgraff and Juntilla, 1979). For dehulled seed, germination was highest (>95%) at temperatures 20°C and lowest (<10%) at 30°C, while hulled seed expressed deeper dormancy at 20°C (Fig. 2), suggesting that hulls play a role in enhancing thermally-induced secondary dormancy.

View larger version (47K): [in this window] [in a new window]   Fig. 2. Germination percentages at 14 d at 15, 20, 25, and 30°C without and with light (16/8 h dark/light) in dormant, hand dehulled (without and with hulls), and hulled seed grown at Kernen and the Seed Farm in 2003. Standard errors are represented by vertical bars. For non-dormant seed lots, germination percentages were 95% (P = 0.05) within 14 d of testing (data not shown), except for hulled samples incubated at 30°C with 8 h light (81% ± 5% germination at both locations).

Germination of dehulled and dehulled (with hulls) seed was similar, regardless of light or temperature (Tables 2, 3, and 4), indicating that the hulls unlikely played a role in supplying chemical inhibitors. The involvement of growth inhibitors from hulls is generally not established in grasses (Simpson, 1990). Further research is needed to elucidate the role, if any, of inhibitors such as abscisic acid, alkaloids, and phenolic acids (Kaur and Singh, 1988) in inducing dormancy in P. canariensis.

View this table: [in this window] [in a new window]   Table 4. Effect of three temperatures regimes, without and with light, on days required to overcome dormancy ( 98% germination) in dormant, hand dehulled and hulled seed grown at two locations in 2003.

Effect of Treatments on Water Uptake in Hand Dehulled Seed
None of the H₂O₂, GA, and ethephon treatments tested had an effect on water uptake in dehulled seed, while H₂O₂ and GA treatments overcame dormancy (Table 2). Juntilla et al. (1978) reported that treatment of P. arundinacea with 1.44 to 2.88 mM GA stimulated germination in dehulled seed to 90%, but only 25% in hulled seed. Despite the effectiveness of GA, its use in Exp. 6 was not attempted as research has associated 0.72 mM GA with etiolated seedling growth in wheat (Matus-Cádiz and Hucl, 2003). Use of H₂O₂ is not recommended in seed testing likely because it is associated with abnormal shoot and root growth (Huang and Hsiao, 1987). In Exp. 6, H₂O₂ and ethephon were not tested as they were associated with abnormal root formation in Exp. 4 and 5.

Effect of Treatments on Water Uptake in Hulled Seed
The hulls appear to play a minor role in restricting water transport to the seed (Table 3). Treatment with H₂O₂ was the most effective treatment, stimulating germination in hulled seed to 72% (SF). In contrast, Junttilla and Nilsen (1980) reported that germination of P. arundinacea (with light) at 27°C was stimulated to only 10% using 0.02 to 1 M H₂O₂. The modes of action of H₂O₂ in the termination of dormancy likely involve (i) the modification or scarification of the hull or seed coat membranes or (ii) the provision of additional O₂ (Huang and Hsiao, 1987). It is currently unclear which mode of action, if not both, is partially overcoming secondary dormancy in P. canariensis. All 12 treatments tested, except heating (Treatment 7), resulted in water uptake rates that were one to 2 d faster in hulled seed relative to Treatment 1 (Table 3). Heating involved desiccating caryopses for 3 d at 30°C and, thus, required several more days of water imbibition to rehydrate the seed and initiate germination.

Low germination percentages were observed for hulled seed using KNO₃ (<16% germinated), chilling (<14%), heating (<2%), GA (<30%), ethephon (<33%), and hull pricking (<16%; Table 3). Junttilla and Nilsen (1980) reported that germination of P. arundinacea (with light) at 27°C was stimulated to 45% using 0.01 M KNO₃. Vose (1962) reported that chilling (3°C for 2 wk), before germinating at 23°C, did not significantly stimulate germination in P. arundinacea. Heating has been tested for overcoming dormancy (92% germinated) in only P. minor (Singh and Dhawan, 1976). The use of GA inhibits (Nakamura, 1962; Juntilla et al., 1978) and promotes (Lugwig, 1971) germination in Phalaris spp. Landgraff and Juntilla (1979) reported that 0.004 M ethephon promoted germination to 38% in P. arundinacea (without light). Vose (1956) reported that hull pricking resulted in 37% germination in a P. aquatica x P. arundinacea hybrid at 25°C (with light) for 21 d. Hull pricking did not significantly improve water uptake at 52 h in P. arundinacea (Vose, 1962).

Dormancy attributed to mechanical constraint of germination by the hulls is not common in grasses (Simpson, 1990). The normal pattern of germination in hulled seed primarily involved the radicle rupturing the pericarp and subsequently the lemma, with the coleoptile forcing its way between the caryopsis and lemma. Normal germination also involved the radicle and coleoptile emerging and pushing the hull completely off the germinating caryopsis. Although a varying degree of mechanical resistance was present, it is unlikely that the hulls play a major role in mechanically restricting germination, considering germination can be enhanced by merely pricking the hull (Table 3). Similar conclusions have been made regarding the role of the hulls in P. arundinacea (Vose, 1962).

Effect of Temperature, Light, Substrata, and Treatments on Germination of Hand Dehulled and Hulled Seed
At 15/25°C (16/8h), only hulled seed displayed low germination percentages during 10 d of testing, regardless of treatment or light regime, suggesting that high temperatures likely led to expression of thermally induced secondary dormancy in hulled seed (Table 4). Hulled samples developed deeper secondary dormancy levels under BB (Treatment 4), indicating that its use may have compounded the already anaerobic germination conditions imposed by the hulls. Treating with KNO₃, heating, or chilling, before germinating at 15/25°C (with or without light), was not required to effectively overcome dormancy in dehulled seed, indicating that dehulling is an effective treatment for rapidly germinating seeds at 15/25°C (without or with light). At 20/30°C, dehulled and hulled seed displayed thermally induced secondary dormancy, regardless of treatment or light regime (Table 4), indicating that the use of 20/30°C (without or with light) should be avoided in seed tests when germinating highly dormant seed.

The lemma and palea are causal agents of dormancy in almost every study of dormancy in grasses (Simpson, 1990); however, dehulling is not recommended for germinating grass seed (ISTA, 1985; CFIA, 1997; AOSA, 2002). Vose (1956) reported that dehulling improved germination to 73% in a P. aquatica x P. arundinacea hybrid when tested at 25°C (with light) for 21 d, suggesting to Vose that the hulls likely played a major role in impeding gas exchange in dormant caryopses. Further research is needed to determine if dehulling is an effective treatment in other Phalaris species.

At 15°C (without or with light), dehulling and KNO₃ (Treatments 1, 2, and 5) effectively overcame dormancy, while heating (Treatment 6) also effectively overcame dormancy in 10 d but only when germinated at 15°C (without light). These treatments have advantages and disadvantages. Dehulling breaks dormancy in 6 d, but it is labor intensive and may damage the embryo, particularly if using mechanical dehulling. Dehulling in P. arundinacea and P. aquatica is unlikely to be of interest to breeders because their small caryopses would probably be difficult to dehull without damaging the embryo. Potassium nitrate or heating treatments require 4 and 7 d longer to overcome dormancy, respectively, indicating that hand dehulling is recommended if seed test duration is a concern. For dormant seed, we suggest that hand dehulling be attempted, before considering KNO₃ or heat treatments, because it will likely lead to the most rapid and satisfactory germination percentages.

Dehulling and heating recommendations in the present study have further improved on our previously reported P. canariensis seed germination protocol (Matus-Cádiz et al., 2001). In addition, the length of the seed test has been shortened from 14 to 10 d. It is important to identify the shortest test period, without loss of precision that can be used to germinate economically important species. Delayed issuing of germination certificates have been reported in pedigreed seed lots of P. tuberosa (Easton and Mullet, 1971), owing to the 21 d seed test recommended by the ISTA. In summary, to overcome dormancy rapidly and effectively in annual canarygrass, we recommend (i) hand dehulling seed, (ii) treating hulled seed with 0.01M KNO₃, or (iii) treating hulled seed with heat (3 d at 30°C) before germinating at 15°C for 10 d in darkness using TB. Final counts should be taken at 10 d. Seed should be air-dried at 23°C, under conditions of low relative humidity, for 7 d before conducting germination tests. We expect these germination methods to be of particular interest to plant breeders, seed producers, and seed testing laboratories, which routinely determine the germination percentages of seed lots.

	ACKNOWLEDGMENTS

 
Appreciation is expressed to Lanette Ehman and Nai-Yee Jay for their technical assistance in the field and laboratory, respectively. Funding for this research was provided by a grant from the Saskatchewan Agriculture Development Fund.

Received for publication June 8, 2004.

	REFERENCES

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