The effects of light, temperature, after-ripening, nitrate and water on Chenopodium album seed germination

.............................................................................................................. iii LITERATURE REVIEW ............................................................................................ 1 MATERIALS AND METHODS .................................................................................. 9 RESULTS ............................................................................................................... 14 DISCUSSION .......................................................................................................... 22 APPENDIX I. SEED COLLECTION SITES ............................................................. 26 APPENDIX II. ADDITIONAL TABLES ..................................................................... 27 REFERENCES CITED ............................................................................................ 33 ACKNOWLEDGMENTS ......................................................................................... 36


Introduction
Chenopodium album (common lambsquarters) is a widespread and troublesome weed in Iowa and agricultural areas throughout the north temperate regions of the world. A considerable number of studies have been published characterizing the seed germination of C. album. Seed germination and seedling emergence from the soil seed pool is a critical threshold life history event immediately preceding assembly in agricultural communities and subsequent interference with crop productivity. The effects of light, temperature, after-ripening, nitrate and moisture level on the germination of C. album are reviewed herein.

Light
Light has been shown to increase germination of Chenopodium album (Baskin and Baskin, 1977;Cumming, 1963;Henson, 1970;Jursik et al., 2003;Karssen, 1967;Moravcova and Dostalek, 1989;Vincent and Roberts, 1977;Roberts and Benjamin, 1979;Wentland, 1965), particularly in combination with nitrate Karssen, 1989, 1993;Henson, 1970;Moravcova and Dostalek, 1989;Roberts and Benjamin, 1979;Wentland, 1965). A light and nitrate interaction is also noticeable when studying the seeds whose parent plants were exposed to nitrate (Saini, Bassi and Spencer, 1985b). However, a few studies such as Williams (1962), have reported no significant difference in germination rates between seeds germinated in the light and in the dark. Karssen (1967) found that responsiveness to a single red irradiation at 23°C following imbibition in darkness increased germination with increasing pre-irradiation time up to hour 16 (90% germination). In a second study by Karssen (1970), the ideal interval between imbibition and irradiation was 24 hours at 23°C and 48 hours at 4°C. Interestingly, imbibed seed exposed to light and subsequently dried, retain light-induced changes (Henson, 1970).

Parent plant photoperiod
C. album response to photoperiod length has been studied intensely at the level of the parent plant and in seed germination tests. Parent plants exposed to long photoperiods (16-17 hours) produce a higher percentage of dormant seeds (Jursik et al., 2003;Wentland, 1965) as well as more seeds overall (Wentland, 1965) than plants exposed to 8 hour photoperiods. Parent plants exposed to long photoperiods produce seeds which require light to germinate and which are stimulated by red or white light and inhibited by far red light. Seeds produced under short days do not require light to germinate and are unaffected by red or far red light (Wentland, 1965). In a unique study, Wentland (1965) found that it is the length of photoperiod and not the intensity of light experienced by the parent plant which determines the dormancy of seeds.

Seed photoperiod
Seeds germinated under long photoperiods or continuous light have lower germination rates compared to seeds germinated under short photoperiods (Cumming, 1959), reportedly taking four times as long to reach 50% germination (Wentland, 1965). This trend of decreasing germination with increasing photoperiod has also been noted in combination with nitrate (Henson, 1970), incandescent lighting (Cumming, 1963) and restricted water supply (Cumming, 1963). Traditional photoperiods are not necessary to produce high rates of germination. Exposure to 15-16 minutes of light once (with nitrate, Henson, 1970) or twice Karssen, 1989, 1993) can be sufficient to obtain high rates of germination. As little as 15 seconds of light was sufficient for older seed at 23°C in a study performed by Henson (1970). Regardless of short or long day germination, seeds germinate at higher percentages in the light when the perianth has been removed (Wentland, 1965).
The timing of imbibition, light exposure and nitrate application plays a large role in germination rates (Karssen, 1967). Henson (1970) discovered that when 16 minute of light was supplied 36 hours following imbibition, the interval between nitrate application and light exposure determined the germination percentage. The highest germination (about 60%) occurred when nitrate was applied from the start of imbibition (36 hour interval between nitrate and light exposure). In the same study, seeds imbibed with nitrate but not exposed to light only reached about 5% germination.
Furthermore, seeds imbibed in darkness for extended amounts of time subsequently required more light energy to stimulate germination (Henson, 1970).

Light x Temperature
There is a noted interaction between light and temperature, with higher rates of germination typically seen with alternating as opposed to constant temperatures in the light (Henson, 1970;Moravcova and Dostalek, 1989;Murdoch, Roberts and Goedert, 1989). Vincent and Roberts (1977) found that light, in combination with alternating temperatures, has a more significant effect when the warmer temperature is maintained for 16 hours rather than 8 hours. Henson (1970) reported that alternating temperatures with a 20°C difference inc reased germination to over 40% whereas a variation of 10°C resulted in only about 5% germina tion.

Light x Temperature x Nitrate
Light, temperature and nitrate interact strongly as characterized by Henson (1970), Moravcova and Dostalek (1989), Vincent and Roberts (1977) and Roberts and Benjamin (1979) (with extended after-ripening time). Henson (1970) found that treatments with light plus nitrate had twice the germination at 23°C than seen at either 10°C or 30°C, and greater germination than any treatment of light or nitrate applied separately at 10, 23, or 30°C.

Nitrate
Application of nitrate to agricultural soils and in the lab have been shown to stimulate the germination of C. album seeds Karssen, 1989, 1993;Fawcett and Slife, 1978;Henson, 1970;Moravcova and Dostalek, 1989;Spencer, 1985a, 1986;Williams, 1962Williams, , 1963Williams and Harper, 1965;Wentland, 1965;Vincent and Roberts, 1977;Roberts and Benjamin, 1979). Endogenous nitrate concentration in seed is directly related to the parent plant's exposure to exogenous nitrate (Fawcett and Slife, 1978;Saini, Bassi and Spencer, 1985b) or exogenous nitrate applied directly to seed (Williams, 1962). Heteromorphic seed from the same parent plant has been shown to contain different endogenous nitrate levels, with brown seed having a higher average endogenous nitrate concentration than black seed (Williams 1962(Williams , 1963. Nitrate application to the parent plant also increases seed production but it does not alter the ratio of brown to black seed produced (Williams, 1962).
Exposure of seeds to exogenous nitrate can result in significantly higher germination within 4 days of application compared to germination with non-supplemented seed (Saini, Bassi and Spencer, 1985a). The parent plant's history of exposure to nitrate can affect how seeds are subsequently stimulated to germinate. Application of exogenous nitrate to seeds from nitrogen-deficient parent plants resulted in lower germination rates compared to seeds produced by nitrogen-rich parent plants (Fawcett and Slife, 1978). The interaction between light, nitrate and age of seed may also be significant. In a study by Henson (1970), nearly all young seed required a combination of light plus nitrate to germinate, whereas 30% of older seed was able to germinate with neither light nor nitrate.
Light and nitrate have a stimulatory effect on C. album seed germination but nitrate alone can be stimulatory. Saini, Bassi and Spencer (1985a) reported higher rates of germination in the dark when seeds were exposed to nitrate. Karssen (1989, 1993) found the same trend, although slight, which became more pronounced in combination with desiccation of seeds.

Nitrate x Temperature
Higher germination rates have been reported in the dark with nitrate at alternating temperature regimes as opposed to with nitrate at constant temperatures (Vincent and Roberts, 1977;Saini, Bassi and Spencer, 1986). Moravcova and Dostalek (1989) showed increased germination when nitrate was supplied to seed in combination with pre-chilling (after-ripening) treatments. Similarly, Williams (1962Williams ( , 1963 found that increased endogenous nitrate levels coincide with longer pre-chilling periods.

Nitrate x After-ripening Time
Response to nitrate and pre-chilling treatments may be specific to seed morphs. Williams and Harper (1965) reported that brown-reticulate and brown-smooth seed have >90% germination without after-ripening (at 5°C) or nitrate application whil e black-reticulate seed show no increase in germination from after-ripening, but do increase germination from 63 to 90% with the addition of nitrate. Black-smooth seed increase germination from 32 to 61% with after-ripening alone, and increase germination from 32 to 94-95% with nitrate (with or without after-ripening). These findings indicate that after-ripening can partially substitute for lack of nitrate, particularly in black-smooth seed.
High concentrations of nitrate such as 0.309 M were not found to be stimulatory (Henson, 1970).

After-ripening
After-ripening (AR) is generally defined as any conditioning a seed undergoes postabscission and is often synonymous with pre-chilling or storage at low temperatures in moist conditions in the dark. AR has been shown to stimulate germination in C. album, particularly in combination with light and nitrate (Moravcova and Dostalek, 1989;Williams, 1962Williams, , 1963Williams and Harper, 1965;Roberts and Benjamin, 1979;Vincent and Roberts, 1977). However, AR has little effect when seeds are subsequently germinated in the dark with no nitrate at constant temperatures (Moravcova and Dostalek, 1989). Williams and Harper (1965) concluded that AR temperatures of -5, 0, and +5°C were equally effective on both wet and dry seed.
With 3 weeks of exposure to 5°C temperatures, Willi ams and Harper (1965) demonstrated that seeds can increase germination percentage from about 30 (no after-ripening) to 65%. Roberts and Benjamin (1979) reported that seeds exposed to constant 30°C conditions in the light reached maximum germination of around 25% when after-ripened for 4 days at 4°C. Comparatively, seeds not after-ripened and kept at a constant 30°C in the li ght had 0% germination. Williams (1963) reported that after-ripening for 3 to 4 weeks is required, in combination with other stimulatory treatments, to maximize germination. Roberts and Benjamin (1979), however, reported the "best chilling treatment" to be 4 days at 4°C, in the dark, without nitrate. Extended AR may result in reduced germination and possibly induction of secondary dormancy (Roberts and Benjamin, 1979), although Williams (1962) found that seeds tested after 12 or 52 weeks of afterripening showed the same germination percentage as after 4 weeks of after-ripening. Roberts and Benjamin (1979) found that after-ripening in the dark results in the ability to better respond to light and nitrate interaction than after-ripening in the light.

Moisture Level
In the majority of published C. album studies, response to moisture level has not been investigated and thus was not quantified or reported. Typically, studies have been carried out on filter paper in petri dishes which are rewetted as needed throughout the experiment. Dry seeds are considered to be insensitive to stimulation by light, although imbibed seeds exposed to light and subsequently dried, retain light-induced changes (Henson, 1970). A combination of continuous light and restricted water (0.5 ml compared to 1.0 ml) inhibited germination as reported by Cumming (1963) in both incandescent and (less so) fluorescent lighting. Bouwmeester and Karssen (1989) reported slightly higher rates of germination from seeds which were desiccated following burial in soil and prior to germination treatments in the light and dark, with nitrate. Chu, Sweet and Ozbun (1978) reported that seeds soaked in running tap water germinated at a higher percentage than unsoaked seed. However, Wentland (1965) found that washing seed for up to 96 hours had no effect on germination at 8 or 17 hour photoperiods.
However, Williams and Harper (1965) reported that brown seed is capable of germination at 0°C with water. Henson (1970) hypothesized that high temperatures may induce thermo-dormancy, in effect reducing seed response to favorable temperatures.

Constant and alternating temperatures
Seeds exposed to light without nitrate and to nitrate without light saw high germination rates at alternating temperatures of 10-30°C (20° differe nce) but not when exposed to alternating temperatures of 15-25°C (10° difference) (Henson,1 970). The highest germination reported by Moravcova and Dostalek (1989) involved a combination of light, nitrate, alternating temperatures, and after-ripening. Even with no after-ripening, alternating temperatures (10-30°C) produced a larger germination response with light, nitrate or light plus nitrate treatments compared to the same treatments under constant 25°C. A similar positive interaction occurred among light, nitrate, alternating temperatures and AR as reported by Vincent and Roberts (1977).

Seed Heteroblasty and Phytochrome
Seed heteroblasty is the variable dormancy-germinability capacity among individual seeds shed by a single parent plant at abscission. Heteroblasty is induced in the seed at the time of embryogenesis by the parent plant and is retained for the life of the seed. This original dormancy-germinability state changes with life history and annual cycling in the soil seed pool. The underlying mechanisms controlling this dynamic state in seeds may be due to the presence of multipleinteracting phytochromes modulating seed behaviors. Further analysis of the phytochrome system is outside the scope of this paper but has been published in several papers Smith, 1975, 1977abc;Morgan and Smith, 1978, 1981Smith, 2000;Smith and Whitelam, 1997;Smith, Casal and Jackson, 1990).

The Goal of This Research
From this literature review, several deficits are evident in our understanding of Chenopodium album germination. For example, the history of seed prior to experimentation may have an effect on the germinability of seed, but this information is often incomplete or missing in published research. A complete seed history should include the following: a) the date of harvest, b) the ecological description of the population habitat, c) seed harvest and storage preparation description, d) storage condition and e) duration of storage pre-experiment. Of these necessary pieces of information, only 4% of the 24 common lambsquarters germination studies reviewed herein included all five details, 29% included four, 25% included three, 25% included two, and 17% included only one or no details of the seed history. This lack of seed history information seriously compromises the repeatability, comparability and interpretation of these published studies.
Another gap in our understanding of C. album germination comes from the limited number of parameters (e.g. light, temperature, after-ripening, nitrate, and water) which have been examined within any one study. Of the 24 studies focused on C. album germination reviewed here, only 12% included five or more parameters, 21% included four, 29% included three, and 38% included only one or two. Additionally, only 4 of the studies compared multiple populations of common lambsquarters.
With these limitations in our understanding of the dynamic nature of Chenopodium album seed germination, we designed a study to determine the combined effects of light, temperature, afterripening time, nitrate and water level and their interactions. We studied two Iowa populations of C. album derived from a common farm in central Iowa and thoroughly detailed the seed history of each population.  Figure 6).

METHODS AND MATERIALS
Immediately after harvest, 2007 and 2008 seed was passively air-dried on screens for 9 and 7 days, respectfully. The perianth and pericarp (see Seed Heteromorphy, below) remained intact on the majority of seed as collected in the field. At no point was the perianth or pericarp forcefully removed. The dried seeds were then placed in 250 to 500 ml translucent HDPE Nalgene containers with screw-tight lids (Fisher Scientific, Pittsburgh, Pennsylvania, USA). The 2007 population was immediately placed into storage at constant temperatures of -20, 4 or 20°C with very low to no light.
The 2008 population was stored at a constant 20°C f or 132 days prior to storage at 4°C (see Table   1a). Seeds were taken from these conditions immediately prior to use in each of the experiments.
The effects of seed storage temperature and duration were accounted for as random effects in the ANOVA performed. However, a closer look at the individual replications for the 2007 population reveals possible germination effects due to storage temperature with seed stored at 20°C having higher germination rates than those 4°C which in t urn had higher rates than those stored at -20°C (See Appendix II Table 1b and Figure 7). Because this storage condition effect was accounted for when constructing the ANOVA and therefore did not largely affect the results, storage condition will not be a primary discussion point in this paper. Differences in the duration of storage for the 2007 and 2008 populations of six and twelve weeks respectively did not have any noticeable effect on germination rates (See Appendix II Table 1b and Figure 7).

Seed Heteromorphy
Chenopodium album germinates spring through late summer with peaks of germination in April and August (Williams, 1963). Flowers are arranged in glomerules, forming spikes at a terminal panicle (Gleason and Cronquist, 1963). Seeds appear to be disk-shaped with two convex sides.
Detailed seed morphology and classification is not agreed upon in published literature, but similarities are evident among descriptions as illustrated herein.
As described by Delorit (1970), seeds of C. album are black, with a glossy surface and enclosed in a semi-translucent granular pericarp. The five-parted perianth may also be attached to the seed, but is often detached during collection and subsequent handling. The seeds of C. album have been described by Williams and Harper (1965) as having four distinct forms at maturity: brownreticulate, brown-smooth, black-reticulate and black-smooth. Additionally, all of these seed types may be present on the same plant. Chu, Sweet and Ozbun (1978) described seed as having four color morphs: brown, deep brown, brown-black and black but they did not distinguish between smooth or reticulate. Wentland (1965) described the variation in color as yellow, orange, and dark red being characteristic of immature seed, whereas mature seeds are dark in color. A few studies considered here (Henson, 1970;Moravcova and Dostalek, 1989) stated that they used only black seed while most studies did not specify seed morphology.
The study reported herein used only seed which appeared to be brown to dark brown in color, with pericarp intact or nearly complete, as this was the state of seed when collected. A perianth was present on roughly half of seeds used, in an open or closed formation, as collected in the field. Immature, yellow, orange or red seeds were omitted from experiments.

Germination
Germination was evaluated in 30 ml gas-tight vials, with 20 mm outside diameter mouths (Wheaton Science Products, Millville, New Jersey, USA) (as described in Dekker and Hargrove, 2002). Two disks of Anchor Blue germination blotter paper (Anchor Paper Co., St. Paul, Minnesota, USA), 32 mm in diameter, were placed in, and completely covered, the bottom of the vials. Following insertion of blotter paper, 0.75, 1.00, or 1.25 ml of distilled, de-ionized water (or KNO 3 solution, as described below) was added to the vials along with ten dry, mature C. album seeds which had been sorted under a dissecting microscope to ensure quality. After placing water solutions and seeds in the vials, they were immediately sealed with neoprene stoppers and crimped (Wheaton hand crimper, model 22430; Wheaton Science Products, Millville, New Jersey, USA) around the vial neck with an aluminum ring to ensure a gas-and watertight seal. All vials were then grouped and wrapped in two layers of aluminum foil (except light treatments with no after-ripening period). Seed to be germinated in the dark remained in foil for the entirety of the experiment. After preparation, vials were placed in either after-ripening (AR 1 to 10 weeks) or germination (AR 0 weeks) assay conditions. Vials remained sealed throughout after-ripening and the germination experiment. Each replication of 396 vials was set up individually within the span of 11 hours, starting from the time seeds were removed from storage and ending when sealed vials were moved into controlled AR or germination cabinets.
All vials were prepared under normal laboratory lighting at room temperature.

After-ripening
Seeds of the 2007 and 2008 populations were exposed to after-ripening (AR) conditions at constant 4°C in the dark, while moist, for 0 to 10 weeks. Darkness was achieved by wrapping vials in two layers of aluminum foil.

Germination assay
The sealed vials with germination paper, water (or KNO 3 ), and seed were placed in one of three Hoffman (model SG-30, Hoffman Manufacturing, Albany, Oregon, USA) controlled environment seed germination cabinets for seven days, after which germination data was collected. Each of the three chambers alternated in twelve hour shifts between a low and high temperature: 5-15°C (cold), 15-25°C (warm) and 25-35°C (hot) with all temperatu res accurate to ± 1°C. In each of these chambers a dark (24 h  ). Brand of tube and assignment to cabinets was not controlled. After seven days in the germination cabinets, the number of seeds germinated was determined for each vial. Germination was evidenced by radicle protrusion outside the seed hull.

Nitrate
Preliminary nitrate assays showed C. album to have the highest germination at 0.01 M KNO 3 compared to 0.0001, 0.001, 0.1, or 0.25 M KNO 3 with no germination occurring at or above 0.5 M KNO 3 (see Appendix II Table 5). As such, two levels of nitrate (0, 0.01 M) were used in the studies reported herein.

Moisture
Preliminary assays showed water levels above 0.5 ml and below 2.0 ml stimulated germination. Levels above 2.0 ml showed a declining germination trend as water levels increased up through 4 ml (see Appendix II Table 6). As such, three levels of distilled water (0.75, 1.00, 1.25 ml) were used in this study.

Experimental Design and Analysis
This study used a factorial arrangement to test all

Population
For the 2007 population, light, temperature, nitrate and after-ripening were significant factors.
Water was not found to be a significant factor. The parameters were examined as three-way interactions: light by temperature by after-ripening, light by nitrate by after-ripening and light by temperature by nitrate. The mean square error for the 2007 population was 0.82. For further ANOVA results, see Appendix II Table 2.

Light x Temperature x After-ripening Time
Light stimulated the 2007 C. album population germination but its effects varied with temperature (temp) and after-ripening duration (4°C , dark, moist; time AR ) prior to germination when averaged over water and nitrate levels ( Fig. 1). All germination percent means have a standard error of 17%.
Dark. Germination in the dark varied from 0 to 1% (temp cold ), 0 to 2% (temp warm ) or remained at 0% (temp hot ) and was similar at all time AR at these temperatures (Fig. 1).
Light: among temperatures within after-ripening time. Light-stimulated germination at 15-25°C (temp warm ) and 25-35°C (temp hot ) were similar at all time AR (Fig. 1). Light-stimulated germination at temp warm was greater at 1-8 weeks and similar at 0 and 9-10 weeks time AR relative to that at 5-15°C (temp cold ). Light-stimulated germination at temp hot was greater at 0-5 and 7 weeks and similar at 6 and 8-10 weeks time AR relative to that at temp cold .
Light: within temperatures among after-ripening times. Light-stimulated germination at temp cold varied from 0 to 1% and was similar at all time AR (Fig. 1). Maximum light-stimulated germination (22-24%) occurred at temp warm after 3-5 weeks time AR . Light-stimulated germination at temp warm increased with 0-3 weeks time AR , then decreased with 3-10 weeks time AR and was never less than 4%. Light-stimulated germination at temp hot was greater at 3 weeks compared to 6 and 8-10 weeks time AR and was never less than 5%. 0 1 2 3 4 5 6 7 8 9 10 Light, Hot Figure 1: The effects of light, temperature and after-ripening time (4°C, dark, moist; time AR ) averaged over water and nitrate levels on 2007 Chenopodium album population seed germination (%). Top figure: germination percent means for warm or hot with (*) were significantly different from cold at that after-ripening time at the p = 0.05% level. Warm and hot were similar at all after-ripening times. Bottom table: germination percent means with the same letter were not significantly different at the p = 0.05% level.

Light x Nitrate x After-ripening Time
Light stimulated the 2007 C. album population germination but its effects depended on the presence of nitrate in the water solution and after-ripening duration prior to germination when averaged over temperature and moisture levels (Fig. 2). All germination percent means have a standard error of 14%.
Dark. Germination in the dark was very low and remained at 0% (water only) or varied from 0 to 1% (0.01 M nitrate) and was similar at all time AR (Fig. 2  Light: among nitrate levels within after-ripening time. Light-stimulated germination with 0.01 M nitrate was greater at 1-8 weeks and similar at 0 and 9-10 weeks time AR relative to that in only water within each of those time AR (Fig. 2).

Light: within nitrate levels among after-ripening times. Light-stimulated germination in
only water varied from 2 to 7% and was similar at all time AR (Fig. 2)

Light x Temperature x Nitrate
Light stimulated the 2007 C. album population germination but its effects vary with temperature and the presence of nitrate in the water solution when averaged over water quantity and after-ripening duration (Fig. 3). All germination percent means have a standard error of 7.1%.
Dark. Germination in the dark was very low and remained at 0% (water only) or varied from 0.1 to 0.7% (0.01 M nitrate) and was similar at all temperatures (Fig. 3). In the dark, no germination occurred in only water at temp warm or temp hot .
Light: among nitrate levels within temperature. Light-stimulated germination with 0.01 M nitrate was greater at temp warm and temp hot and similar at temp cold relative to that in only water within each of these temperatures (Fig. 3). Light: within nitrate levels among temperatures. Light-stimulated germination in only water varied from 0.1 to 6.1% and was similar at temp warm and temp hot , both of which were greater than that at temp cold (Fig. 3). Maximum light-stimulated germination (21.1%) occurred with 0.01 M nitrate at temp warm . Light-stimulated germination with 0.01 M nitrate at temp warm was greater than that at both temp cold and temp hot , while that at temp hot was greater than at temp cold .

Population
For the 2008 population, light, temperature, nitrate, after-ripening and water were significant.
These parameters were examined as four-way interactions: light by temperature by nitrate by water and light by temperature by nitrate by after-ripening. The mean square error for the 2008 population was 0.62. For further ANOVA results, see Appendix II Table 2.

Light x Temperature x Nitrate x Water
Light stimulated the 2008 C. album population germination but its effects varied with temperature, nitrate and water quantity when averaged over after-ripening duration (Fig. 4). All germination percent means have a standard error of 14%.
Dark. Germination in the dark was very low and varied from 0 to 1% and was similar at all temperatures, nitrate and water quantities (Fig. 4).
Light: among water and nitrate levels within a temperature. Light-stimulated germination at temp cold remained at 0% and was similar with and without the presence of nitrate at all water quantities (Fig. 4). Within both temp warm and temp hot , light-stimulated germination in the presence of nitrate was similar with the highest water quantities (1.25, 1.00 ml) and both were greater than that at 0.75 ml water. In the absence of nitrate, light-stimulated germination within temp warm and temp hot were similar at all water quantities. Within both temp warm and temp hot , light-stimulated germination in the presence of 0.01 M nitrate was greater than that in only water at all water quantities.
Light: within water and nitrate levels among temperatures. In the presence of nitrate and light, germination at temp warm was greater than that at both temp cold and temp hot , while that at temp hot was greater than that at temp cold (Fig. 4). In the absence of nitrate, light-stimulated germination at temp warm was greater than that at both temp cold and temp hot for all water quantities. Light-stimulated germination in the absence of nitrate was similar at temp cold and temp hot for all water levels.

Light x Temperature x Nitrate x After-ripening Time
Light stimulated the 2008 C. album population germination but its effects varied with temperature, presence of nitrate and duration of after-ripening time when averaged over water quantity (Fig. 5). All germination percent means have a standard error of 7.5%.
Dark. Germination in the dark was very low and varied from 0 to 1.3% and was similar at all temperatures, nitrate levels and after-ripening durations (Fig. 5).
Light: among temperature and nitrate levels within after-ripening time. Light-stimulated germination at temp warm with 0.01 M nitrate was greater than that at all other conditions when compared at each time AR from 1-9 weeks, with maximum germination (50.7%) at week 5 (Fig. 5) Figure 5: The effects of light, temperature, nitrate and after-ripening time averaged over water quantities on 2008 Chenopodium album population seed germination (%). Top figure: germination percent means were significantly different from all other means (*); those with (+) were significantly different from cold and hot with only water and cold with nitrate; and those with (^) were significantly different from cold with and without nitrate; all criteria within an after-ripening time at the p = 0.05% level. Bottom
In the absence of light, germination was not different from zero regardless of nitrate application.
These results did not agree with the findings of Saini, Bassi and Spencer (1985a) whom reported higher rates of germination in the dark when seeds were exposed to nitrate. Here, in light with no nitrate, germination occurred at moderate rates and the addition of nitrate stimulated germination further. This illustrated that the combination of light plus nitrate is more effective than either treatment applied individually.
C. album seeds after-ripened in the dark at cool temperatures and in moist conditions have been reported to germinate at a higher rate, particularly when after-ripening is followed by germination in the light with the application of nitrate (Moravcova and Dostalek, 1989;Williams, 1962Williams, , 1963Williams and Harper, 1965;Roberts and Benjamin, 1979;Vincent and Roberts, 1977). These reports were supported by the results of this experiment, where after-ripening at 4°C in the dark in moist conditions for approximately 3-5 weeks in the light with nitrate (and at warm temperatures) was the most stimulatory of all conditions investigated. Roberts and Benjamin (1979) postulated that extended after-ripening times may result in reduced germination or induction of secondary dormancy.
This hypothesis may explain the decrease in germination rates seen after 7, 8, 9 and 10 weeks of after-ripening in the light with nitrate and/or warm to hot temperatures (Fig. 1, 2 and 5). However, Williams (1962) found that C. album seeds after-ripened for both 12 and 52 weeks had similar germination to those after-ripened for 4 weeks, essentially demonstrating no decrease in germination due to extended after-ripening. Future C. album germination studies looking at the effects of extended after-ripening times at one-week intervals would provide further insight into this matter.
The effects of moisture on C. album germination have not been widely examined in previous reports. Based on preliminary studies (see Appendix II table 6) and those found herein, there seems to be an optimum range of moisture levels which stimulate germination. Having examined levels ranging from 0.5 to 4.0 ml, the optimum water level appears to be approximately 1.25 to 2.0 ml for the vial system used in these experiments (see methods and materials for more information on the vial system).
It was apparent in this study that cold temperatures (5-15°C, alternating in 12 hour cycles) inhibit germination regardless of light, nitrate and after-ripening (all of which factors would otherwise be providing stimulatory germination effects). Warm (15-25°C) and hot (35-35°C) temperatures were effective at promoting germination in the light, especially with the addition of nitrate and 3-5 weeks of after-ripening. This positive interaction among light, nitrate, alternating temperatures and afterripening was also reported by Vincent and Roberts (1977). The optimal (constant) temperature range for germination of C. album is reportedly between 15 and 25°C which agreed with this study which found higher germination rates with alternating temperatures 15-25°C relative to 5-15°C and 25-35°C.

Population similarities
Cold

Future Directions
The overarching goal of this project is to build a condensed C. album seed germination assay to characterize individual populations to predict seedling emergence patterns in agricultural soils in an effort to decrease interference with crop productivity. The study presented herein is the foundation of this research. The next step will be to examine C. album populations from locations outside of the Midwest to determine if the range of factors examined here are also sufficiently stimulatory for other populations. Once a suitable range of light, temperature, nitrate, water and after-ripening times have been established, the study would progress to analyzing populations collected from agricultural fields in a condensed seed germination assay in an effort to characterize the C. album seedling emergence pattern. The results of this analysis would then be used to better control weed populations by applying herbicides as the most opportune time based on light exposure, temperatures, duration the seed spent in moist soil at cool temperatures, and nitrate and water levels found in the soil from whence the population was collected.      1.0000 0.00% 1 Averaged over light, temperature and after-ripening time (0, 2, 4 weeks); 2007 population. Seed stored 238 days at 4°C 2 Averaged over light, temperature and after-ripening time (0, 2, 4 weeks); 2007 population. Seed stored 294 days at 4°C 3 Averaged over light, temperature, after-ripening time (0, 2, 4 weeks) and water (0.5, 0.75, 1.0 ml); 2007 population. Seed stored 343-427 days at -20°C 1.00% 1 Averaged over light, temperature and after-ripening time (0, 2, 4 weeks); 2007 population. Seed stored 133 days at 20°C 2 Averaged over light, temperature and after-ripening time (0, 2, 4 weeks); 2007 population. Seed stored 287 days at 4°C 3 Averaged over light, temperature, after-ripening time (0, 2, 4 weeks) and nitrate (0, 0.001, 0.01 M); 2007 population. Seed stored 343-427 days at -20°C