Seed and Bulb production fromMechanically 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

•Long storage life

(approx. 10 years)•Takes 3 years from

seed to seed

•Seedlings fragile

Seed

Dormant bulbs

•Short storage life

(approx. 6 months)•Flowers first year

after sewing

•Once in soil can

survive draught

Experimental Design:

Propagule = dormant bulbs with 7 - 15 mm diameter

Randomized complete block w/ 4 reps per treatment

Treatments:

Hand planted bulbs

1inch between-plant spacing2 inch between-plant spacing

Mechanically planted bulbs

1inch between-plant spacing2 inch between-plant spacing

Two year trial- 2009 and 2010

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

Emergence

Flowers/umbel

Seed/plant

Bulbs/plot

(2009)

DATA COLLECTED:

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

year

planting

method spacing emergence

avg.

flowers/

umbel

avg.

seed/plant

avg. total

seed

weight(g)

avg. total

no. seeds

avg. 100

seed

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.

planting

method

between

bulb

spacing

avg. total

bulbs

avg. no.

bulbs 7-

15 mm

avg. no.

bulbs

<7mm

avg. no.

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 PavelkaCorey Wahl

Emily Gibson

Saber Jewel

Thank you for listening!

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 f ire damage, invasive exotic annual grasses and noxious weeds, anthropogenic development, recreation, livestock overgrazing, and mining. The Great Basin Native Plant Selection and Increas e 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 . 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 quantif ied by the presence of key indicator species ( Johnson and Braun 1999). The presence of wild onion ( Allium spp.) and other key shrubs and forbs are associated with good rearing habitat

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 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 a large range compared to many of the North American native Alliums (Hellier 2000). Although many species of wild Alliumsgrow throughout the Great Basin, A. acuminatum was selected due to its wide range and association with quality sage grouse habitat. Genetic diversity will be analyzed using Sequence Related Amplif ied 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-situconservation and germplasm for ex situ conservation. The in -situ conservation will be complementary to ongoing ex-situconservation at the Western Regional Plant Introduction Station.

Diversity of Allium acuminatum in the Great BasinRobert Adair, RC Johnson, Barbara Hellier and Walter Kaiser

USDA-ARS Western Regional Plant Introduction Station Pullman, Washington

Methods

Possible collection site locations were obtained from a variety of sources including specimens at the University of Nevada at Reno herbarium, data provided by contacts at herbaria in Oregon, Idaho and Nevada, f ield observations by US Forest Service and Bur eauof Land Management personnel, and collection data from a preliminary f ield study in 2004. Information was organized into a spreadsheet for f ield use and entered into a GIS-based map to aid in collection planning. The GIS data from Omernick Ecoregions

and our possible collection sites were joined in order to identify A. acuminatum populations located in unique ecoregions (Minami 2000). This procedure allowed us to prioritize collection sites and maximize the probability of collecting plants that may h aveadapted to special or rare environments. Level III and IV Omernick Ecoregions geospatial data is available for most of the conterminous United States on the Environmental Protection Agency website (EPA 2005).

Bulb size and morphology was recorded and then planted in the greenhouse in root -trainer containers with a ¾ new soil and ¼ perlite mixture. In November 2006, planted bulbs were placed in a vernalization chamber at 4˚C and watered bi-monthly. In the

spring, viable plants will be transplanted to f ield plots at Pullman and Central Ferry for common garden studies involving measurement of various phenotypic characteristics ( i.e. f lower and anther color, leaf length and width, leaf number, scape length and diameter) . Tissue samples were collected from plants growing in the vernalization chamber and DNA extractractions were carried out using the Promega Wizard kit for plant DNA. DNA extractions were quantif ied by f lourometry and used as template fora modif ied SRAP-PCR protocol using 7 infrared dye labeled primer sets. Amplif ied PCR products were run on the LICOR DNA analysis system.

To examine the feasibility of using bulked DNA from populations for genetic analysis, a preliminary analysis was run using in dividuals

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 bulkedtissue 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.

Results

The collection area spanned 1430 m (4692 ft) of elevation and covered approximately 620 km (385 mi.) east -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) , and assigned a shape description (Figure 4) . The bulbs were then stored at the Western Regional Plant

Introduction Station in a temperature-controlled room at 15 C. A total of 3,107 A. acuminatum bulbs were sampled throughout Idaho, Oregon and Nevada.

Average bulb diameter was 1.0 cm with a standard deviation of 0.2 cm for the entire collection. The maximum bulb diameter was 2.1 cm and minimum diameter was 0.3 cm, and the maximum and minimum 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

classif ied as f lat globe, and 1.9% high-globe.

Analysis of bulb diameter at the 55 collections sites indicated a signif icant dif ference among collection sites (F= 10.2, p < 0.05) , however, this analysis cannot separate the specif ic environmental and genetic factors related to bulb size. Despite statistical dif ferences, the general uniformity in bulb size and morphology was striking given the large area and diversity of environmental conditions from which samples were collected.

Collection site coordinates were entered into a GIS database and a 30 -meter radius from the A. acuminatum

population was compared to Level III and IV ecoregions as described by Omernick (1987). This analysis showed A. acuminatum populations were collected from 20 Level IV ecoregions (Table 1) . Dissected High Lava Plateau was the most common ecoregion representing 24% of the 55 sites.

The results of our preliminary SRAP indicate that bulking tissue from populations can show differences in genetic variation at least in the two populations that were selected for testing. These results also indicate that SRAP markers detect genetic variations between individuals of a given population (Figure 4) .

Conclusion/ Future Directions

The use of GIS information allowed for ef f icient planning and organization of our f ieldwork. Maps that were created identifying possible collection sites provided a visual aid for route planning. GIS data such as ecoregions and land status (ownership) were helpful in determining priority and appropriateness of collection sites.

Although having coordinates for possible collection sites was generally useful, this was limited by the quality of the sourcedata. Herbarium specimen data identifying sites were at times over 40 years old, and habitat change can make them unreliable. Changes in land status were 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 f ield, and therefore BLM Surface Management maps (1:100,000) were consulted for increased resolution and ease of use.

Analysis of SRAP produced molecular markers from two populations, Old Penitentiary (#27) and Road to 3 Mile Creek (#33) , indicated that

dif ferences in genetic variation can be determined using both individual plant DNA extraction and bulks of tissue from populations. We are currently working in the lab to run a bulked sample of 20 randomly selected individuals f rom each population to characterize genetic dif ferences among the 55 populations collected.

These data along with measurements of genotypic variation from our common garden studies will be used to identify key populations for in-situ and ex-situ conservation sites and to delineate seed transfer zones for future restoration projects.

Acknowledgements

This research is part of the Great Basin Native Plant Selection and Increase Project. Funding was provided by a grant from theUSDI BLM 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 acuminatumf ieldwork. We also thank Lisa Taylor, Allan Brown, and Ted Kisha for their laboratory assistance.

References

[EPA] Environmental Protection Agency. 2005. Level IV Ecoregions. URL: http://www.epa.gov/wed/pages/ecoregions/level_iv.htm Corvallis (OR) Western Ecology Divis ion.

H ellier, B. C. 2000. G enetic, Morphologic, and H abitat Divers ity of Two Species of Allium Native to the Pacific Northwest, USA and Their Implications for In Situ Seed Collection for the National

Germplasm System. (MSc. Thesis ) Pullman, WA. Washington State University.

Johnson, H . K. and C. E. Braun. 1999. Viability and Conservation of an Exploited Sage G rouse Population. Conservation Biology. 13 (1): 77-84.

Li, G . and C. F. Quiros . 2001. Sequence Related Amplified Polymorphisms (SRAP), a new markers system based on a s imple PCR reaction: it’s application

to mapping and gene tagging in Brass ica. Theoretical and Applied Genetics . 103:455-461.

Miller, R. F., and L. L. Eddleman. 2001, Spatial and temporal changes of Sage Grouse habitat in the sagebrush biome: Oregon S tate Univers ity, Agricultural Experiment Station, Technical Bulletin 151, Corvallis , OR.

Minami, Michael. 2000. Us ing ArcMap. Redlands (CA): Environmental Research Institute, Inc. Press . 528 p.

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.

[USDA NRCS] USDA Natural Resources Conservation Service. 2005. The PLANTS database. Vers ion 3.5. URL: http://plants .usda.gov/ Baton Rouge (LA). National Plant Data Center

[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 Mountain Research Station.

[USDI BLM] Bureau of Land Management. 2005 G reat Basin Restoration initiative. URL: http://www.fire.blm.gov/gbri/ index.html (accessed 29 August 2005) Boise (ID): BLM Office of Fire and Aviation

Level 4 Ecoregion Collection sites

Semiarid Hills and Low Mountains 1

Southern Forested Mtns/Dry Partly Wooded Mtns 1

Mountain Home Uplands 1

Southern Forested Mountains 1

Pluvial Lake Basins 1

High Desert Wetlands 1

Continental Zone Foothills 1

Unwooded Alkaline Foothills 1

Semiarid Foothills 1

High Glacial Drift-Filled Valleys 1

Central Nevada Mid-Slope Woodland and Brushland 1

Central Nevada High Valleys 1

Carbonate Woodland Zone 2

Carbonate Sagebrush Valleys 2

Mid-Elevation Ruby Mountains 3

Semiarid Uplands 4

High Lava Plains 5

Upper Humboldt Plains 6

Owyhee Uplands and Canyons 8

South of collection s ite at Martin Creek guard s tation (H umbolt County NV).

Large A. acuminatum population near Buchanan (H arney Co. OR) Pink areas on the hills ideare flowering A. acuminatum.

Figure 1. A. acuminatum collected at Roland Road(Owyhee Co. ID) showing umbels , scapes, and bulbs.

Figure 4. Bulb morphology. (A) Single, globed (B) cloved (C) Three-cloved(D) H igh-globed (E) Flat-globed.

Table 1. Number of A. acuminatum populations collected within Level IVOmernick Ecoregions (Omernick 1987).

Figure 3. Dis tribution of A. acuminatum collection s ites across the G reat Basin.

Figure 2. Example of a section of SRAP gel electrophores is of A. acuminatum DNA us ing em1-me2 primers (Li and Quiros 2001). Bulked samples from Old Penitentiary s ite (#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.

Figure 4. Dendrogram of individual and bulked samples from two populations ofA. acuminatum constructed by unweighted pair group method with arithmetic averaging

based on s imple matching coeffficients of SRAP results .

A. acuminatum population in Owyhee County, Idaho.