Seed and Bulb production from Mechanically Planted Allium acuminatum Bulbs

Barbara Hellier & Richard Johnson USDA-ARS Western Regional Plant Introduction Station, Pullman, WA Allium propagules for establishing crop or plants in a landscape Seed •Long storage life (approx. 10 years) •Takes 3 years from seed to seed •Seedlings fragile

Dormant bulbs •Short storage life (approx. 6 months) •Flowers first year after sewing •Once in soil can survive draught Two year trial- 2009 and 2010

Propagule = dormant bulbs with 7 - 15 mm diameter Experimental Design: Randomized complete block w/ 4 reps per treatment Treatments: Hand planted bulbs 1inch between-plant spacing 2 inch between-plant spacing Mechanically planted bulbs 1inch between-plant spacing 2 inch between-plant spacing Field prep preplant: Plots rototilled for easy hand planting No fertilizer

Planter: Hage Belt Cone Planter

60 bulbs per plot

All bulbs planted at depth of 1 inch

No irrigation DATA COLLECTED: Emergence

Flowers/umbel

Seed/plant

Bulbs/plot (2009) Allium acuminatum umbel with open and immature capsules. Summary of emergence, flowers/umbel, seed harvested/plant, total seed weight, total no. seeds and 100 seed weight data from 25 randomly selected plants per plot for 2009 & 2010

avg. avg. total avg. 100 planting flowers/ avg. seed avg. total seed year method spacing emergence umbel seed/plant weight(g) no. seeds weight(g) 2009 hand 1inch 55.5 30.8 31.1 1.5 778.0 0.19 2009 hand 2inch 57.0 28.9 37.2 2.0 929.0 0.22 2009 mechanical 1inch 56.0 29.5 39.0 1.9 975.5 0.20 2009 mechanical 2inch 56.5 30.9 34.5 1.8 860.8 0.20 2010 hand 1inch 51.8 36.8 33.1 1.7 828.0 0.20 2010 hand 2inch 50.3 34.6 30.2 1.6 756.5 0.21 2010 mechanical 1inch 47.8 39.4 41.8 2.2 1043.5 0.21 2010 mechanical 2inch 52.0 37.0 34.7 1.8 866.3 0.21 Summary of bulb production from the Allium acuminatum seed production trial 2009.

between avg. no. avg. no. planting bulb avg. total bulbs 7- bulbs avg. no. method spacing bulbs 15 mm <7mm bad bulbs hand 1 inch 94.0 64 b* 4.3 25.8 hand 2 inch 103.5 79 ab 1.3 23.8 mechanical 1 inch 104.2 81 a 1.3 21.8 mechanical 2 inch 104.2 72 ab 1.0 31.0 *Means sharing letters are not different using the LSD at P<0.05. Production problems

Disease: Mostly disease free (so far) 2010 Rust outbreak (Rust was seen in wild populations in ID) Didn’t affect seed production in 2010. Weeds Shattering Summary: Mechanical bulb planting can successfully be used for seed production.

There was no statistical difference in seed production for plots with one and two inch between bulb spacing.

Between bulb spacing of two inches produced a higher number of 7-15mm diameter bulbs. Allium acuminatum seed increase plot, Pullman, WA. Thank you’s and Acknowledgements Great Basin Native Plant Selection and Increase Project Nancy Shaw Richard Johnson Rob Adair

My Crew: Marie Pavelka Corey Wahl Emily Gibson Saber Jewel Thank you for listening!

Diversity of Allium acuminatum in the Great Basin Robert Adair, RC Johnson, Barbara Hellier and Walter Kaiser USDA-ARS Western Regional Plant Introduction Station Pullman, Washington

Large A. acuminatum population near Buchanan (Harney Co. OR) Pink areas on the hillside are flowering A. acuminatum. Background

Rangeland restoration is increasingly important to improve habitat quality in arid western areas that have been severely degraded due to multiple factors including widespread fire damage, invasive exotic annual grasses and noxious weeds, anthropogenic development, recreation, livestock overgrazing, and mining. The Great Basin Native Plant Selection and Increase Project (NPSIP) represents a multi-organizational collaboration between federal and state agencies, universities and private companies to investigate and apply new techniques of range management to promote a healthy ecosystem (USFS-RMRS 2005).

The continued threats to ecosystem health in the Great Basin rangelands have led to the loss of both plant and animal habitat . South of collection site at Martin Creek guard station (Humbolt County NV). For example, Greater sage grouse (Centrocercus urophasianus Bonaparte) currently occupy approximately 56% of suitable habitat available before the rapid settlement by people of European descent (BLM 2005). Survival and fecundity rates for sage grouse may depend on habitat quality, which is commonly quantified by the presence of key indicator species (Johnson and Figure 4. Dendrogram of individual and bulked samples from two populations of Braun 1999). The presence of wild onion ( Allium spp.) and other key shrubs and forbs are associated with good rearing habitat A. acuminatum constructed by unweighted pair group method with arithmetic averaging based on simple matching coeffficients of SRAP results. for sage grouse(Miller and Eddleman 2001).

For this project we chose to collect and maintain Allium acuminatum Hook. (Alliaceae) for purposes of biodiversity conservation Conclusion/ Future Directions and potential future use in rangeland reclamation and restoration projects. A. acuminatum is a perennial herb native to North America which grows throughout most of the Western States and Western Canada (Figure 1) (USDA 2005). A. acuminatum has The use of GIS information allowed for efficient planning and organization of our fieldwork. Maps that were created identifyingpossible a large range compared to many of the North American native Alliums (Hellier 2000). Although many species of wild Alliums collection sites provided a visual aid for route planning. GIS data such as ecoregions and land status (ownership) were helpful in Figure 3. Distribution of A. acuminatum collection sites across the Great Basin. grow throughout the Great Basin, A. acuminatum was selected due to its wide range and association with quality sage grouse determining priority and appropriateness of collection sites. habitat. Genetic diversity will be analyzed using Sequence Related Amplified Polymorphisms (SRAPS) and these data along with additional information from common garden studies will be used to delineate seed transfer zones and choose sites for in-situ Although having coordinates for possible collection sites was generally useful, this was limited by the quality of the sourcedata. Herbarium conservation and germplasm for ex situ conservation. The in-situ conservation will be complementary to ongoing ex-situ specimen data identifying sites were at times over 40 years old, and habitat change can make them unreliable. Changes in land status were Results conservation at the Western Regional Plant Introduction Station. also important in our study since our objective was to collect mainly from BLM and USFS land. Determining land status from GIS created maps or software was not always feasible in the field, and therefore BLM Surface Management maps (1:100,000) were consulted orf The collection area spanned 1430 m (4692 ft) of elevation and covered approximately 620 km (385 mi.) east - increased resolution and ease of use. west and 445 km (277 mi.) north-south, between N 39 to 44 latitude and W 114º to 119º longitude in the Western United States (Figure 3). The bulbs from the 55 collection sites were counted, measured (diameter), Analysis of SRAP produced molecular markers from two populations, Old Penitentiary (#27) and Road to 3 Mile Creek (#33), indicated that and assigned a shape description (Figure 4). The bulbs were then stored at the Western Regional Plant differences in genetic variation can be determined using both individual plant DNA extraction and bulks of tissue from populations. We are Introduction Station in a temperature-controlled room at 15 C. A total of 3,107 A. acuminatum bulbs were currently working in the lab to run a bulked sample of 20 randomly selected individuals from each population to characterizegen etic sampled throughout Idaho, Oregon and Nevada. differences among the 55 populations collected.

Average bulb diameter was 1.0 cm with a standard deviation of 0.2 cm for the entire collection. The These data along with measurements of genotypic variation from our common garden studies will be used to identify key populations for maximum bulb diameter was 2.1 cm and minimum diameter was 0.3 cm, and the maximum and minimum in-situ and ex-situ conservation sites and to delineate seed transfer zones for future restoration projects. site means were 1.2 and 0.9 cm, respectively. Of the bulbs collected, 91% were single bulbs, 8.7% were cloved bulbs, and 0.2% were 3-cloved bulbs. Most of the bulbs were globed shaped (90.3%), 6.8% were Figure 4. Bulb morphology. (A) Single, globed (B) cloved (C) Three-cloved classified as flat globe, and 1.9% high-globe. (D) High-globed (E) Flat-globed. Analysis of bulb diameter at the 55 collections sites indicated a significant difference among collection sites Level 4 Ecoregion Collection sites (F= 10.2, p < 0.05), however, this analysis cannot separate the specific environmental and genetic factors Semiarid Hills and Low Mountains 1 related to bulb size. Despite statistical differences, the general uniformity in bulb size and morphology was Southern Forested Mtns/Dry Partly Wooded Mtns 1 striking given the large area and diversity of environmental conditions from which samples were collected. Mountain Home Uplands 1 Southern Forested Mountains 1 Collection site coordinates were entered into a GIS database and a 30 -meter radius from the A. acuminatum Pluvial Lake Basins 1 population was compared to Level III and IV ecoregions as described by Omernick (1987). This analysis High Desert Wetlands 1 showed A. acuminatum populations were collected from 20 Level IV ecoregions (Table 1). Dissected High Continental Zone Foothills 1 Lava Plateau was the most common ecoregion representing 24% of the 55 sites. Unwooded Alkaline Foothills 1 Semiarid Foothills 1 The results of our preliminary SRAP indicate that bulking tissue from populations can show differences in High Glacial Drift-Filled Valleys 1 genetic variation at least in the two populations that were selected for testing. These results also indicate Central Nevada Mid-Slope Woodland and Brushland 1 that SRAP markers detect genetic variations between individuals of a given population (Figure 4). Figure 1. A. acuminatum collected at Roland Road Central Nevada High Valleys 1 (Owyhee Co. ID) showing umbels, scapes, and bulbs. Carbonate Woodland Zone 2 Carbonate Sagebrush Valleys 2 Mid-Elevation Ruby Mountains 3 Semiarid Uplands 4 A. acuminatum population in Owyhee County, Idaho. High Lava Plains 5 Acknowledgements Upper Humboldt Plains 6 Owyhee Uplands and Canyons 8 This research is part of the Great Basin Native Plant Selection and Increase Project. Funding was provided by a grant from theUSDI BLM Methods Table 1. Number of A. acuminatum populations collected within Level IV Omernick Ecoregions (Omernick 1987). Great Basin Restoration Initiative through the USDA Forest Service Rocky Mountain Research Station. We would like to thank Ch eri Howell (USFS), Jean Findley (BLM), Ann Debolt (USFS), Lynn Kinter (USFS), and Nancy Shaw (USFS) for providing location data for Allium acuminatum Possible collection site locations were obtained from a variety of sources including specimens at the University of Nevada at Reno fieldwork. We also thank Lisa Taylor, Allan Brown, and Ted Kisha for their laboratory assistance. herbarium, data provided by contacts at herbaria in Oregon, Idaho and Nevada, field observations by US Forest Service and Bur eau References of Land Management personnel, and collection data from a preliminary field study in 2004. Information was organized into a [EPA] Environmental Protection Agency. 2005. Level IV Ecoregions. URL: http://www.epa.gov/wed/pages/ecoregions/level_iv.htm Corvallis (OR) Western Ecology Division. spreadsheet for field use and entered into a GIS-based map to aid in collection planning. The GIS data from Omernick Ecoregions Hellier, B. C. 2000. Genetic, Morphologic, and Habitat Diversity of Two Species of Allium Native to the Pacific Northwest, USA and Their Implications for In Situ Seed Collection for the National and our possible collection sites were joined in order to identify A. acuminatum populations located in unique ecoregions (Minami Germplasm System. (MSc. Thesis) Pullman, WA. Washington State University. 2000). This procedure allowed us to prioritize collection sites and maximize the probability of collecting plants that may h ave adapted to special or rare environments. Level III and IV Omernick Ecoregions geospatial data is available for most of the Johnson, H. K. and C. E. Braun. 1999. Viability and Conservation of an Exploited Sage Grouse Population. Conservation Biology. 13 (1): 77-84. conterminous United States on the Environmental Protection Agency website (EPA 2005). Li, G. and C. F. Quiros. 2001. Sequence Related Amplified Polymorphisms (SRAP), a new markers system based on a simple PCR reaction: it’s application to mapping and gene tagging in Brassica. Theoretical and Applied Genetics. 103:455-461. Bulb size and morphology was recorded and then planted in the greenhouse in root -trainer containers with a ¾ new soil and ¼ Miller, R. F., and L. L. Eddleman. 2001, Spatial and temporal changes of Sage Grouse habitat in the sagebrush biome: Oregon S tate University, Agricultural Experiment Station, Technical Bulletin perlite mixture. In November 2006, planted bulbs were placed in a vernalization chamber at 4˚C and watered bi-monthly. In the 151, Corvallis, OR. spring, viable plants will be transplanted to field plots at Pullman and Central Ferry for common garden studies involving Minami, Michael. 2000. Using ArcMap. Redlands (CA): Environmental Research Institute, Inc. Press.528 p. measurement of various phenotypic characteristics (i.e. flower and anther color, leaf length and width, leaf number, scape length Omernik, J.M., 1987, Ecoregions of the conterminous United States (map supplement): Annals of the Association of American Geographers, v. 77, no. 1, p. 118-125, scale 1:7,500,000. and diameter). Tissue samples were collected from plants growing in the vernalization chamber and DNA extractractions were [USDA NRCS] USDA Natural Resources Conservation Service. 2005. The PLANTS database. Version 3.5. URL: http://plants.usda.gov/ Baton Rouge (LA). National Plant Data Center carried out using the Promega Wizard kit for plant DNA. DNA extractions were quantified by flourometry and used as template for a modified SRAP-PCR protocol using 7 infrared dye labeled primer sets. Amplified PCR products were run on the LICOR DNA analysis [USDA-FS RMRS] USDA Forest Service. 2005. Great Basin Native Plant Selection and Increase project. URL: http://www.fs.fed.us/rm/boise/research/shrub/greatbasin.html Boise (ID): Rocky Figure 2. Example of a section of SRAP gel electrophoresis of A. acuminatum DNA using em1-me2 primers Mountain Research Station. system. (Li and Quiros 2001). Bulked samples from Old Penitentiary site (#27) are highlighted in in purple, and bulks from Rd. to 3 Mile Creek are in Brown. Individual plant DNA extractions are to the left of each respective bulk sample. [USDI BLM] Bureau of Land Management. 2005 Great Basin Restoration initiative. URL: http://www.fire.blm.gov/gbri/index.html (accessed 29 August 2005) Boise (ID): BLM Office of Fire and To examine the feasibility of using bulked DNA from populations for genetic analysis, a preliminary analysis was run using in dividuals Aviation from a collection site near the Old Penitentiary (#27) in the Snake River plain ecoregion in Idaho, and another site on the r oad to Three Mile Creek (#33) in the Northern Basin and Range ecoregion in Nevada. For this analysis 20 individuals, as well as bulked tissue samples of 5, 10, 15, and 20 plants were run on a LICOR gel electrophoresis system (Figure 2). Gel images were printed out and handscored based on 75 markers from 7 primer sets and analyzed using NTSYS statistical software.