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
year
planting
method
spacing
2009
2009
2009
2009
2010
2010
2010
2010
hand
hand
mechanical
mechanical
hand
hand
mechanical
mechanical
1inch
2inch
1inch
2inch
1inch
2inch
1inch
2inch
avg.
avg. total
avg. 100
flowers/ avg.
seed
avg. total seed
emergence umbel seed/plant weight(g) no. seeds weight(g)
55.5
57.0
56.0
56.5
51.8
50.3
47.8
52.0
30.8
28.9
29.5
30.9
36.8
34.6
39.4
37.0
31.1
37.2
39.0
34.5
33.1
30.2
41.8
34.7
1.5
2.0
1.9
1.8
1.7
1.6
2.2
1.8
778.0
929.0
975.5
860.8
828.0
756.5
1043.5
866.3
0.19
0.22
0.20
0.20
0.20
0.21
0.21
0.21
Summary of bulb production from the Allium
acuminatum seed production trial 2009.
planting
method
hand
hand
mechanical
mechanical
between
bulb
spacing
1 inch
2 inch
1 inch
2 inch
avg. no.
avg. total bulbs 7bulbs
15 mm
94.0
64 b*
103.5
79 ab
104.2
81 a
104.2
72 ab
avg. no.
bulbs
<7mm
4.3
1.3
1.3
1.0
*Means sharing letters are not different using the LSD at P<0.05.
avg. no.
bad bulbs
25.8
23.8
21.8
31.0
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 (H arney Co. OR) Pink areas onthe hills ide
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 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 quantified 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 Alliums
grow 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 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
conservation and germplasm for ex situ conservation. The in -situ conservation will be complementary to ongoing ex-situ
conservation at the Western Regional Plant Introduction Station.
South of collection s ite at Martin Creek guard s tation (H umbolt County N V).
Figure 4. Dendrogram of individual and bulked s amples from two populations of
A. acuminatum cons tructed by unweighted pair group method with arithmetic averaging
bas ed on s imple matching coeffficients of SRAP res ults .
Conclusion/ Future Dire ctions
Figure 3. Dis tribution of A. acuminatum collection s ites acros s the G reat Basin.
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.
Figure 4. Bulb morphology. (A) Single, globed (B) cloved (C) Three-cloved
(D) H igh-globed (E) Flat-globed.
Level 4 Ecor egion
Figure 1. A. acuminatum collected at Roland Road
(Owyhee Co. ID) s howing umbels , s capes, and bulbs.
Methods
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
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
classified as flat globe, and 1.9% high -globe.
Analysis of SRAP produced molecular markers from two populations, Old Penitentiary (#27) and Road to 3 Mile Creek (#33), indicated that
dif f erences in genetic variation can be determined using both individual plant DNA extraction and bulks of tissue f rom popula
t ions. We are
currently working in the lab to run a bulked sample of 20 randomly selected individuals f rom each population to characterizegenetic
differences among the 55 populations collected.
These data along with measurements of genotypic variation f rom our common garden studies will be used to identif y key populations f or
in-situ and ex-situ conservation sites and to delineate seed transf er zones f or f uture restoration projects.
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).
Acknowledgements
A. acuminatum population in Owyhee County, Idaho.
This research is part of the Great Basin Native Plant Selection and Increase Project. Funding was provided by a grant from the USDI 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 acuminatum
fieldwork. We also thank Lisa Taylor, Allan Brown, and Ted Kisha for their laboratory assistance.
References
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, field observations by US Forest Service and Bur eau
of Land Management personnel, and collection data from a preliminary field study in 2004. Information was organized into a
spreadsheet for field 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 ave
adapted 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).
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 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.
Although having coordinates f or possible collection sites was generally usef ul, this was limited by the quality of the sourcedata. Herbarium
specimen data identif ying 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 sof tware was not always f easible in the f ield, and theref ore BLM Surf ace Management maps ( 1:100,000) were consulted of r
increased resolution and ease of use.
Analysis of bulb diameter at the 55 collections sites indicated a significant difference among collection sites
(F= 10.2, p < 0.05), however, this analysis cannot separate the specific environmental and genetic factors
related to bulb size. Despite statistical differences, the general uniformity in bulb size and morphology was
striking given the large area and diversity of environmental conditions from which samples were collected.
Owyhee Uplands and Canyons
8
Table 1. N umber of A. acuminatum populations collected within LevelIV
Omernick Ecoregions (Omernick 1987).
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 field plots at Pullman and Central Ferry for common garden studies involving
measurement of various phenotypic characteristics (i.e. flower 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 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
system.
The use of GIS inf ormation allowed f or ef f icient planning and organization of our f ieldwork. Maps that were created identif iyng possible
collection sites provided a visual aid f or route planning. GIS data such as ecoregions and land status ( ownership) were helpf ul in
determining priority and appropriateness of collection sites.
[EPA] Environmental Protection Agency. 2005. Level IV Ecoregions. U RL: http:/ / www.epa.gov/wed/ pages /ecoregions /level_iv.htm Corvallis (OR) W estern Ecology Divis ion.
H ellier, B. C. 2000. G enetic, Morphologic, andH abitat Divers ity of Two Species of Allium N ative to the Pacific Northwes t, U SA and Their Implications for In Situ Seed Collection for the N ational
G ermplas m Sys tem. (MSc. Thesis ) Pullman, WA. Was hington State University.
Johns on, H . K. and C. E. Braun. 1999. Viability and Conservation of an Exploited SageG rous ePopulation. Cons ervation Biology. 13 (1): 77-84.
Li, G . and C. F. Quiros . 2001. Sequence Related Amplified Polymorphis ms (SRAP), anew markers sys tem bas ed on as implePCR reaction: it’s application
to mapping and gene tagging in Bras s ica. Theoretical and Applied G enetics . 103:455-461.
Miller, R. F., and L. L. Eddleman. 2001, Spatial and temporal changes of Sage Grous ehabitat in the s agebrus h biome: Oregon S tate Univers ity, Agricultural Experiment Station, Technical Bulletin
151, Corvallis , OR.
Minami, Michael. 2000. U s ing ArcMap. Redlands (CA): Environmental Res earch Ins titute, Inc. Press .528 p.
Omernik, J.M., 1987, Ecoregions of the conterminous U nited States (map s upplement): Annals of the As sociation of American Geographers ,v. 77,no. 1, p. 118-125,s cale 1:7,500,000.
[U SDA N RCS] U SDA N atural Res ources Cons ervation Service. 2005. The PLAN TS databas e.Vers ion 3.5. URL: http:/ / plants .usda.gov/ Baton Rouge (LA). National Plant Data Center
Figure 2. Example of a s ection of SRAP gel electrophores is of A. acuminatum DN A us ingem1-me2 primers
(Li and Quiros 2001). Bulked s amples from OldPenitentiary s ite (#27) are highlighted in in purple, and bulks from
Rd. to 3 Mile Creek are in Brown. Individual plant DN A extractions are to the left of each res pectivebulk sample.
[U SDA-FS RMRS] U SDA Fores t Service. 2005. Great Basin Native Plant Selection and Increas e project. U RL:http:/ / www.fs .fed.us /rm/ bois e/res earch/ shrub/ greatbasin.html Bois e (ID): Rocky
Mountain Res earch Station.
[U SDI BLM] Bureau of Land Management. 2005 G reat Basin Restoration initiative. U RL: http:/ / www.fire.blm.gov/gbri/ index.html (acces sed 29 August 2005) Boise (ID): BLM Officeof Fire and
Aviation