Authors name, title, address, contact information

October, 2019 FINAL STUDY REPORT Aberdeen Plant Materials Center

Aberdeen, Idaho

Germination response of curlycup gumweed seed to oxygenated water treatment

Derek Tilley, Terron Pickett

ABSTRACT Seed germination in an oxygenated water bath has been shown to be effective at reducing or eliminating dormancy barriers of some native western North American plant species; however, its full utility is not known. Particularly lacking is information on oxygenated water treatments on native forbs with physiological dormancy. Curlycup gumweed [Grindelia squarrosa (Pursh) Dunal] is a commonly occurring, early-seral forb, native to arid and semi-arid habitats in the Intermountain West used in wildlife habitat and other rangeland planting practices. We compared germination rates and final germination percentage of curlycup gumweed collections from 5 locations in the Intermountain Region exposed to 4 germination treatments: 1) oxygenated water bath, 2) non-oxygenated water bath, 3) 90-d cold/moist stratification, and 4) a non-treated control. Germination rates and final germination percentages after 35-d in oxygenated bath treatments were equal to or greater than those from the 90-d stratification for all 5 accessions. Seed priming in oxygenated water treatments appears to be feasible for quickly and uniformly germinating seed in a laboratory environment with the possibly of expanding the technique for field settings.

INTRODUCTION

Seed enhancement technologies have been viewed as a possibility to improve seedling germination success in rangeland seedings in the Intermountain Western sagebrush biome (Madsen and others 2016; Madsen and others 2018; Anderson and others 2019). Often, fall dormant seedings result in unintentional early germination because of unexpected warm spells in late-fall (James and others 2011) resulting in high numbers of seedling mortality from frost damage. Seed priming could be used to initiate quick germination for planting during periods of suitable moisture and temperatures while allowing adequate time for seedlings to achieve the minimum required growth to survive through winter. Seed priming may be especially useful in post-fire situations where immediate establishment is necessary to prevent erosion during winter and the following spring (Beyers 2004; USDA-NRCS 2013). Seed dormancy mechanisms, common among forb species in the Intermountain West, however, complicate the issue, as seeds require extended stratification periods prior to germination. Determining seed germination requirements and identifying dormancy traits can provide valuable information leading to improved field establishment (Kildisheva and others 2018). Additionally, treatments to quickly and easily overcome seed dormancy mechanisms can facilitate laboratory germination and greenhouse propagation where uniform germination is desirable.

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It has been shown that some species with significant stratification periods respond positively to submersion in oxygenated water (Young and Young 1986; Liu and others 2012; Tilley 2013). For example, Tilley and others (2013) showed that oxygenated water treatments were able to overcome physiological dormancy mechanisms and increase final germination percentages in Nebraska sedge (Carex nebrascensis Dewey), a species of stream margins and wetlands, yet it is unclear how feasible this treatment is for use on upland forbs that may be maladapted to periods of saturation.

Curlycup gumweed is a native, short-lived perennial forb adapted to disturbed upland areas throughout North America except for the southeastern states. It is currently under investigation for use in upland restoration plantings in the Great Basin where it occurs in disturbed areas and roadsides competing with introduced Eurasian annual grasses and forbs (Tilley and Pickett 2019). Curlycup gumweed is highly attractive to native bees and has potential for inclusion in pollinator friendly habitat plantings (Figure 1). Data from the USDA-Agriculture Research Station Bee Research Laboratory in Logan, UT indicate visitations by species from over 40 genera of bees to curlycup gumweed (Ikerd 2016), while Lee-Mäder and others (2016) state it is mostly visited by leafcutter bees (Megachile spp.), long-horned bees (Melissodes spp.), and green metallic sweat bees (Agapostemon spp.). Its drought tolerance and late-season flowering make it especially promising for use in Conservation Reserve Program (CRP) and other range plantings in the arid west where late-blooming forbs are limited (Ogle and others 2011).

Curlycup gumweed may also have value as a food source for at-risk upland birds. Peterson (1970) documented curlycup gumweed as a food source for sage-grouse (Centrocercus urophasianus). In that study, twenty-eight percent of monitored sage-grouse chicks in central Montana between 5 and 8 weeks of age were reported eating curlycup gumweed which made up 3 percent of the crop contents by volume. It was further found to be used by 39 percent of 9 to 12-week-old chicks and made up 4 percent of crop contents of birds of that age group (Peterson 1970).

Numerous reports indicate that curlycup gumweed exhibits physiological dormancy and requires 60 to 90 days of cold-moist stratification for adequate germination (Nuzzo, 1978; Baskin and Baskin 2002; Luna 2008). Neupane and others (2016) observed uneven field germination of spring-seeded curlycup gumweed in studies for biofuel production. The authors reported that seed germination was only accomplished with extensive irrigation, causing field germination attempts to be abandoned and replaced with greenhouse-grown materials. However, fall-dormant seeding of curlycup gumweed in field trials under arid, non-irrigated conditions at Aberdeen,

Figure 1. Curlycup gumweed is a short-lived perennial forb native to western North America with potential for use in conservation and pollinator friendly plantings in arid rangelands.

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Idaho, in which seed could naturally stratify over winter, resulted in essentially 100% establishment of 25 accessions originating from across the Great Basin (Tilley and Pickett 2019).

MATERIALS AND METHODS

We tested germination of five accessions of curlycup gumweed chosen to represent a broad spatial and ecological distribution within the semi-arid west (Figure 2) with four seed treatments to determine if germination rates could be improved without the extended stratification period. Seed collections were made in San Juan County, UT (UT1), Box Elder County, UT (UT2), Park County, WY (WY), Humboldt County, NV (NV1), and Elko County, NV (NV2) (Table 1). Treatments included: 1) oxygenated water bath (air), 2) non-aerated water bath (H2O), 3) 90-d cold-moist stratification (90-d), and 4) non-treated control (control). All seed was of the current season (<1 yr old). Tetrazolium Chloride (TZ) tests were performed by an independent seed lab on each collection to determine potential viability.

Table 1. Collection site location, elevation, soil and associated plant community information for the five evaluated accessions of curlycup gumweed. State and Designation County Lat/Lon Site Characteristics (Web Soil Survey 2018) Elev. (m) NV1 Humboldt 41.1663667, -

117.6821667 Atriplex canescens (Pursh) Nutt., Artemisia tridentata ssp. wyomingensis Beetle & Young, Bromus tectorum L. Enko very fine sandy loam. 20-25 cm precipitation.

1370

NV2 Elko 41.9909167, -114.6701500

Agropyron cristatum (L.) Gaertn., Artemisia tridentata ssp. tridentata Nutt. Chuska-Jackpot-Soughe association. 20-25 cm precipitationx

1610

UT1 San Juan 38.0730028, -109.2464222

Sarcobatus vermiculatus (Hook.) Torr., Helianthus annuus L., Bromus tectorum, Agropyron cristatum, Achnatherum hymenoides (Roem. & Schult.) Barkworth, Sphaeralcea coccinea (Nutt.) Rydb., Salsola tragus L. Mido loamy fine sand. 20-30 cm precipitation.

1780

UT2 Box Elder 41.97631, -112.19040

Bromus tectorum, Artemisia tridentata ssp. wyomingensis, Thinopyrum ponticum (Podb.) z.-W. Liu & R.-C. Wang. Abella gravelly loam. 20-36 cm precipitation.

1360

WY Park 44.976879, -110.699130

Artemisia tridentata Nutt., Pseudoroegneria spicata (Pursh) Á. Löve. Web Soil Survey data not available. Calcareous outcropping.

1930

Figure 2. Location of the five tested accessions of curlycup gumweed.

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For the oxygenated water treatment, we placed 100 seeds in a fine mesh bag and submerged the seed into a mason jar filled with distilled water with an aquarium bubbler for aeration. The non-aerated water treatment was the same, minus the bubbler. Aeration was performed using a Profile 1500 aquarium air pump fitted with a 2.5 cm bubbling air stone (Figure 3). The cold-moist stratification was done by placing seed in a fine mesh bag surrounded by moist potting soil in a 1.1° C (34° F) cooler for 90 days. For stratification and non-treated control treatments, 100 seeds were placed on blotter paper moistened with 5 ml distilled water in 90 mm petri dishes, and the dishes were put in Ziploc bags to retain moisture (Figure 4). All were placed in a Hoffman growth chamber (Hoffman Manufacturing, Inc., Corvallis, Oregon) with a 12-hour light/dark cycle with 22° C day and 15° C night temperatures. Germination was recorded at 7, 14, 21, and 28, and 35 days after initiation (DAI). Plants were considered germinated if they were observed to have an emerging root or shoot greater than 2 mm in length.

Germination rate was determined using the method described by Maguire (1962). The number of seedlings obtained at each counting (7, 14, 21, 28, and 35-DAI) was divided by the number of days after planting, and the values obtained at each count were summed at the end of the test as follows.

Germination rate = (Number of seedlings/days after planting) + … (Number of seedlings/days after planting)

Each treatment was replicated 4 times in a randomized complete block design with each shelf of the growth chamber serving as a block. Final germination at 35-DAI, and germination rates were analyzed with the analysis of variance procedure after the data was determined to be normally distributed using the Shapiro-Wilk normality test using Statistix 10 (Analytical Software, Tallahassee, Florida). Means were separated using the least significant difference test at P<0.05.

Figure 3. Curlycup gumweed seed germinating under oxygenated water conditions. The mesh bag used in the experimentation has been removed to allow viewing.

Figure 4. Curlycup gumweed seed germinated following 90-d cold/moist stratification and non-treated control.

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RESULTS AND DISCUSSION Germination Rates Significant differences in germination rates were observed between treatments of each accession (NV1 p=0.0000, NV2 p=0.0001, UT1 p=0.0000, UT2 p=0.0000, WY p=0.0000). Germination rates of the air treatment were significantly greater than the 90-d stratification treatment for NV1 and UT2 accessions in which air treatments resulted in as much as 2 times higher germination rates (NV1). Germination rates for the air treatment and 90-d stratification treatment were greater than the H2O and control treatments for all accessions and ranged from 3 to 10 times faster than H2O soaking treatments and 2 to 13 times faster than the non-treated control of all five accessions (Table 2). The lowest germination rates were obtained from the H2O treatment in 4 of 5 accessions, while the non-treated control produced the lowest germination rate from the Box Elder County, UT accession (UT2). Table 2. Germination rates of 5 curlycup gumweed accessions using 4 germination treatments: 90-d cold-moist stratification (90-d), oxygenated water bath (Air), non-oxygenated water bath (H2O), and non-treated control (control).

NV1 NV2 UT1 UT2 WY ----------------------------------germination rate-------------------------------------- Air 18.1a1/ 13.5a 9.9a 11.3a 15.7a 90-d 9.1b 12.0a 9.6a 9.1b 14.8a H2O 2.8c 3.8b 1.4c 1.1c 3.3b Control 4.9c 6.3b 2.8b 0.9c 4.6b Mean 8.7 8.9 5.9 5.6 9.6

1/ Letters represent significant differences at the P=0.05 level. Germination Over Time Germination responses to the H2O soaking treatment were very similar to the non-treated control, as both treatments resulted in low germination rates and final germination percentages (Figures 5-9). Oxygenated treatments of the five accessions produced significantly faster germ rates and final germination after 35-d than control and H2O. We first observed radical emergence from the air treatment after only 24-h, and approximately 50 to 80% of the final germinants had emerged from the oxygenated treatments by 7-DAI. After 7-DAI germination leveled to a much slower rate, and seeds continued to more sporadically germinate through day 35. The 90-d cold-moist stratification treatment resulted in a quick burst of early germination closely in line with the oxygenated treatment, however after the initial germination very few additional germinants emerged, resulting in significantly lower final germination percentages.

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Figures 3-9. Graphs indicate germination over time from 0 to 35 DAI of 5 curlycup gumweed accessions from semi-arid sites in the Western United States following 1 of 4 seed treatments including: 1) oxygenated water bath (air), 2) non-aerated water bath (H2O), 3) 90-d cold-moist stratification (90-d), and 4) non-treated control (control).

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Final Germination Laboratory TZ tests indicated viability of the 5 accessions ranging from 96 to 98%. Final germination percentages under our 4 treatments ranged from 5 to 72%, and significant differences were observed among all accessions (NV1 p=0.0000, NV2 p=0.0008, WY p=0.0000, UT1 p=0.0000, UT2 p=0.0000) (Table 3). Seed subjected to the air treatment had significantly greater final germination percentages than those from all other treatments for all five accessions. Final germination percentages of the air treatment ranged from 1.3 to 2.4 times greater than those of the 90-d stratification, 2 to 7 times greater than those of the H2O soaking treatment and 2 to 10 times greater than those in the non-treated control group. Table 3. Percentage of germinated seed after 35d following 4 germination treatments: 90-d cold-moist stratification (90-d), oxygenated water bath (Air), non-oxygenated water bath (H2O), and non-treated control (control).

NV1 NV2 UT1 UT2 WY ----------------------------------------%------------------------------------------ Air 72a1/ 52a 44a 50a 60a 90-d 30b 40b 33b 30b 47b H20 18bc 28c 6c 9c 21c Control 20c 24c 12c 5c 19c Mean 35 36 24 23 37

1/ Letters represent significant differences at the P=0.05 level.

CONCLUSION While not adapted to flooded or saturated water conditions, curlycup gumweed collections from multiple locations in the Great Basin showed an improved germination response from soaking in oxygenated water compared to both the non-stratified control and the water soaking treatments in both germination rates and final germination percentage. Faster germination rates may be useful in quickly capturing available moisture and establishing an adequate taproot before the next dry period, resulting in greater final establishment. Treatment in oxygenated water significantly increased germination rates and percentages over the 90-d of cold-moist stratification, thus eliminating the need for extensive pre-treatments to achieve desired germination. The oxygenated water treatment however failed to achieve full germination potential as determined from TZ tests after 35-d in any of the five tested accessions indicating that dormancy was not fully overcome for at least some portion of the seed. Screening of these techniques on additional upland forbs is needed to ascertain the full extent of its utility. Using oxygenated water treatments to overcome physiological dormancy may have application in laboratory and field settings. The techniques used in this experiment do not require specialized equipment or training and can be utilized by non-research-oriented restoration practitioners and technicians. Oxygenated baths could be a useful method to produce germinants for greenhouse production of certain species, especially in situations where production of plants of a similar age is desirable. Furthermore, field establishment via hydroseeding of seed primed in oxygenated water treatments could be a quick means of establishing plants, when waiting for natural breaking of dormancy mechanisms, such as after wildfire, would be prohibitive (Tilley and St John 2013). Variations in priming requirements of conservation plant species need to be better understood for this practice to be successful.

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