1. Referee Summary AOSA 2016 Meeting Portland, Oregon
GERMINATION PROTOCOL FOR THREE COOL-SEASON NATIVE GRASSES: ELYMUS ELYMOIDES SUBSPECIES BREVIFOLIUS “Subspecies C” (SSP C), ELYMUS MACROURUS (ELMA7), AND ELYMUS WAWAWAIENSIS (ELWA2) Paula M. Moore MS in Agriculture, Research Thesis
Referee Summary
AOSA 2016 Meeting
Portland, Oregon

2. Todays Topics Introduction Current published AOSA protocol
Project Goal and Hypothesis
Background: Why Elymus species?
Research Design and Methodology
Results
AOSA New Rules Proposal
Discussion
References Cited
Acknowledgements

3. Current AOSA Protocols for Elymus
Kind of seed
Substrate a
Temp (oC)
Days first count
Days final count
Specific requirements and notes
Fresh and dormant seed
Elymus Canadensis P
15-30 7 21
Light
Prechill at 5oC for 2 wk
 Elymus cinereus
See Leymus cinereus
15-25 10
 21
 Ungerminated seeds see sec. 6.2f and 6.9m
Elymus elymoides
P,B
15; 20 14
Ungerminated seeds see sec. 6.2f and 6.9m
 Elymus junceus
See Psathrostachys juncea
20-30 5
 14
Light 
Prechill at 5 or 10°C for 5 days
Elymus smithii
See Pascoyrum smithii
B, P, T  7
 28
Dark 
 KNO3 or soil. Ungerminated seeds: see sec. 6.2f and 6.9m
Elymus trachycaulus subsp. trachycaulus
P, TB
Prechill at 5o or 10oC for 5 d. If still dormant on 10th day of germination period, rechill 2 d then place at oC for 4 d
Anonymous. 2014a. AOSA.

4. Project Goal and Hypothesis
The goal of this research was to develop standardized germination protocols for three species of Elymus: SSPC, ELMA7, and ELWA2.
It is hypothesized that Elymus elymoides subsp. brevifolius “SSP. C” will have the same germination protocol as Elymus elymoides subsp. Brevifolius (ELELB2), which is currently published by AOSA (Anonymous, 2014a). Temperatures for optimal germination will be 15oC or 20oC, blotter paper will be the substrate and water the moisture source (Anonymous, 2014a).
Elymus macrourus (Turcz. ex Steud.) Tzvelev, POACEAE, will have a germination protocol with optimal viability at a constant temperature < 25oC with an 8 h light source.
Elymus wawawaiensis (J. Carlson & Barkworth), POAECEA, Snake River wheatgrass will have an optimal viability at a constant temperature < 25oC.
It is hypothesized that no dormancy breaking techniques will be necessary for any of these species, as members of the genus Elymus do not usually require such treatments. Light will be necessary for the germination of all three species.
Statistical evaluation was based on a 95% Confidence Level (P < 0.05).

5. Back Ground: Why Elymus species?
Many uses of Elymus
Ethnobotanical History
Relative of Wheat Family
Great Basin Restoration Project
Economic Importance
Production of Certified Seed Fields in Washington
Reclamation and Restoration Uses
Introduce the Plant Species: photographs

6. Elymus elymoides ssp. brevifolius “SSP C” (SSP C)
(a) Moore, Paula [Photo] Elymus elymoides ssp brevifolius “SSP C”, USDA-ARS, Agriculture Experiment Station of 13 Western States, Washington State University, Central Ferry, Washington.
(b) Moore, Paula [Photo] Elymus elymoides ssp brevifolius “SSP C”, Sample 3, PI , Regenerated at Agriculture Experiment Station of 13 Western States, Washington State University, Central Ferry, Washington.
(c) Granite Seed Company [Photo] Elymus elymoides subspecies brevifolius “Subspecies C” (SSP C, Squirreltail), Lehi, Utah (accessed 26 September 2015)

7. Elymus macrourus (ELMA7)
(a) Moore, Paula [Photo] Elymus macrourus, USDA-ARS, Agriculture Experiment Station of 13 Western States, Washington State University, Central Ferry, Washington.
(b) Moore, Paula [Photo] Elymus macrourus, Sample 4, sfd-11-11pmc148, Regenerated at Palmer Material Center, Alaska.
(c) Moore, Paula [Photo] Elymus macrourus, USDA-ARS, Agriculture Experiment Station of 13 Western States, Washington State University, Central Ferry, Washington.

8. Elymus wawawaiensis (ELWA2)
(a) Moore, Paula [Photo] Elymus wawawaiensis, USDA-ARS, Agriculture Experiment Station of 13 Western States, Washington State University, Central Ferry, Washington.
(b) Moore, Paula [Photo] Elymus wawawaiensis, Sample 5, PI , Regenerated at Central Ferry, Washington.
(c) Moore, Paula [Photo] Elymus wawawaiensis, USDA-ARS, Agriculture Experiment Station of 13 Western States, Washington State University, Central Ferry, Washington.

9. Research Design and Methodology
Preliminary Experiment
Split-plot nested design, temperature as the main plot and accession and seed source as subplots.
Plot treatments were constant temperatures of 15oC, 20oC, and 25oC .
The 432 samples (8 runs x 18 germination boxes x 3 chambers) had 431 degrees of freedom.
An additional 9th run without lights was done at the end.
Germination box placement was randomized for each run with the use of an online tool (
For each sample, 50 seed were counted and placed in 5 rows of 10 seed on top of the moist blotter paper.
A total of 1.65 g of wetable powdered Captan 50% fungicide (Bonide Captan 50% WP Fungicide for Ornamentals and Fruit) was sprinkled over the seed.

10. Continued…
Forceps were dipped in Waxie Green Neutral Cleaner® between samples to reduce the spread of seed coat-borne fungus.
Moisture in the germination boxes was monitored daily by the “thumb indent test” for the moisture content of the blotter paper (Anonymous, 2014b, Section 6.3).
Distilled water was added as necessary to maintain a moist blotter paper.
Seedlings were evaluated on day seven (7 d), fourteen (14 d), twenty- four (24 d) and thirty-five (35 d) for normal seedlings, abnormal seedlings and dead seed.
Testing was continued for an additional two days (2 d) if more than 10% of the imbibed seeds had not germinated on 35 d.
Dead seed were removed from the moist blotter as needed at every evaluation and recorded.
Results for Normal seedlings, Abnormal seedlings, dormant and dead seed were recorded on an excel spread sheet
ELELB2 was utilized as a check, we know from the published protocol how this species should react to temperatures.

11. i Western Regional Plant Introduction Station [WRPIS].
Plant symbol
Sample Id #
Cultivar or Name
Source
Year
ELMA7 1 PI
WRPIS, Pullman, WAi
1991 2
sfd-09-11pmc12
PMC, Palmer, AKii
2009 4
sfd-07-07pmc115
PMC, Palmer, AK
2007 5
sgo-05-05PMC138
2005 6
sfd-11-11pmc148
2011
ELWA2 7
PI , ‘Secar’ 
WRPIS, Pullman, WA
1982 8
PI , D&S-211
1988 9
‘Discovery’
USDA –FRRL, Logan, UTiii 10
USDA –FRRL, Logan, UT 11
‘Secar’
1995 12
2003
SSP C 13
 ‘Grandview’
2000 14
PI , T-1202
2004 15
PI , T-1206
ELELB2 16
‘Tusas’
PMC, Los Lumus, NMii
2008 17
PMC, Los Lumus, NM 18
2006
i Western Regional Plant Introduction Station [WRPIS].
ii Plant Materials Center [PMC].
iii USDA, Forest and Range Research Laboratory [FRRL].

12. Seed Analysis Seed Purity and Pure Seed Unit (PSU)
Samples for the AOSA referee study were identified and analyzed for pure seed unit (PSU), #21 definition of AOSA Rules for Testing Seed (Anonymous, 2014b).
The best fits the morphological structures of Elymus species.
Seed analysts’ at the Washington State Department of Agriculture (WSDA) Seed Program used AOSA approved methods for purity analysis (Anonymous, 2014b).
A total of 450 PSU were analyzed and counted by hand and placed into individual envelopes for distribution to referee participating laboratories.
Seed Working Weights
A total of 16 replicates of100 PSU were counted, weighed and recorded for each seed lot.
The mathematical equations for variance, standard deviation, and coefficient of variance were applied as directed in Section 13.4.d (Anonymous, 2014b).
Data was used to estimate the minimum and maximum weights for Noxious Weed Exam, Purity Exam, and Working Weight Sample.
Every species published in the AOSA Rules for Testing Seeds has a PSU definition and working weights.

13. Research Design and Methodology
Referee Study
A referee panel was established with four to seven laboratories. The referee for SSP C had four participants. The ELMA7 had five participating laboratories. The ELWA2 referee had seven participating laboratories.
Split-plot, nested design with laboratory as the main plot and replicated samples as the subplot.
Labs collected data on the number of seedlings counted as normal germination by AOSA published standards (Anonymous. 2014b).
The main parameter tested was number of normal seedlings on 7 d, 14 d, and 21 d among laboratories.
The seed sources for the referee study included one fresh seed sample that was less than one year old. The fresh seed lots of SSP C and ELWA2 were regenerated from seed stock during the growing season at the USDA-ARS, Central Ferry Research Farm.
The regeneration harvest yielded no pure live seed of ELMA7. The harvest was primarily of inflorescence with immature seed. Therefore, no fresh seed of ELMA7 was available for the referee study.

14. Continued…
The referee study procedures started with saturation of standard blotter paper with distilled water. A single saturated blotter paper was placed in a germination box.
For each provided sample or seed source, a minimum of four replicates were placed in germination boxes and labeled for each species, sample number and replicate.
The chamber temperature was established at a constant 20oC. Chamber temperatures were monitored to assure they were within +/-2oC of the targeted parameter.
All germination boxes were monitored for the moisture content of the blotter paper.
If seeds were determined as ungerminated by 21 d, they were held over for an additional 2 d of testing per AOSA rules (Anonymous, 2014b).
Following a 23-day-test period, any remaining ungerminated seed were Tetrazolium tested (TZ) and calculated TZ viable results were added to the normal seedlings to get total viability.

15. i Western Regional Plant Introduction Station [WRPIS].
Plant symbol
Sample Id #
Cultivar or Name
Source
Year
SSP C 1 PI
WRPIS, Pullman, WAi
2003 2
USDA-ARS, Central Ferry Research Farm, WA
2014 3
‘Grandview’
USDA – FRRL, Logan, UTii
2000
ELMA7
sgo-05-05pmc138
PMC, Palmer, AKiii
2005
sfd-07-07PMC115
PMC, Palmer, AK
2007
sfd-09-11pmc12
2011 4
sfd-11-11pmc148
ELWA2
‘Secar’
USDA – FRRL, Logan, UT
1995
‘Discovery’
PI , ‘Secar’
2013 5 PI 6
i Western Regional Plant Introduction Station [WRPIS].
ii USDA, Forest and Range Research Laboratory [FRRL].
iii Plant Materials Center [PMC].

16. Results

17. Preliminary Experiment Results
(P=0.51) (P=0.08) (P=0.00) (P=0.02) (P=0.10) (P=0.00) (P=0.09) (P=0.10)

18. (P=0.40) (P=0.38) (P=0.43) (P=0.36)

19. (P=0.02) (P=0.17) (P=0.00) (P=0.13)

20. * No significant difference between temperatures 15OC and 20OC for ELELB2, SSP C, and ELWA2 (P= 0.01).
** No significant difference in all three temperature treatments for ELMA7 (P = .67).

21. Referee Study Results
The question still remaining was, when should the evaluation period end?
To assess this, a paired analysis (t-test) was performed on the data provided by the referee participants.
Clarification on the data for referee study:
Normal seedlings were the number counted at each day (14 d and 21 d)
Viability on 14 d was a percentage of the normal seedlings
Viability on 21 d was a percentage of normal seedlings PLUS the TZ viable
Statistical analysis to identify if 21 d evaluation period was necessary.

22. Referee Summary (P < 0.05) SSP C ELMA7 ELWA2
Number of Referee Laboratories 4 5 7
Paired Analysis 14 d - 21 d (paired t-test)
Norm. replicates 14 d
34.50%
7.36%
63.89%
Norm. replicates 21 d
35.88%
14.06%
65.16%
Norm. pooled labs 95% Conf. Interv.
1.39%
5.28%
0.44%
Viability % replicates 14 d
24.50%
Viability % replicates 21 d
Viability % pooled labs 95% Conf. Interv.
2.75% (P=0.05)
11.13% (P=0.02)
0.69% (P=0.00)
Univariate Analysis of Difference
Viability % pooled labs 7 d
different (P=0.0)
Viability % pooled labs 14 d
Viability % pooled labs 21 d
different (P=0.01)
same (P=0.10)
Viability % pooled replicate (lab) 7 d
same (P=0.48)
same (P=0.47)
same (P=0.20)
Viability % pooled replicate (lab) 14 d
same (P=0.37)
same (P=0.46)
same (P=0.70)
Viability % pooled replicate (lab) 21 d
same (P=0.50)
same (P=0.53)
same (P=0.08)
Viability % pooled samples 7 d
Viability % pooled samples 14 d
Viability % pooled samples 21 d
different (P=0.02)
Viability % lab*sample 7 d
Viability % lab*sample 14 d
Viability % lab*sample 21 d

23. AOSA New Rules Proposal
A new AOSA rule proposal was submitted electronically on 15 Oct to the AOSA New Rules Committee.
SSP C: 20C, first count 7 d, final count 14 d, 8 h light.
ELMA7: 20C, first count 7 d, final count 21 d, 8 h light.
ELWA2: 20C, first count 7 d, final count 14 d, 8 h light.
A blind committee panel comprised of ten AOSA members reviewed the proposal and supporting evidence.
The Committee rejected the proposal for SSP C, indicating the AOSA Rules for Testing Seeds were not intended to accommodate the subspecies level. It was recommended to discard the SSP C information and edit the proposal for the final rule proposal to the general membership.
The raw data and analysis in support of the requirement of light was requested. A statistical analysis of the germination data without light was not performed. One run of the experiment was conducted without lights. This limited data would not provide sufficient degrees of freedom for a comparative analysis. The use of 8 h standard light was dropped from the rule proposal verbiage.
The rule proposal was edited as suggested and resubmitted on 31 Jan to the AOSA Rules Committee Chair.

24. Sample Definitions Working Weights 21 Yes Elymus macrourus
Pure seed unit #
Chaffy seed
Kind of seed
Minimum weight for Purity Analysis (grams)
Minimum weight for Noxious-weed seed or bulk examination (grams)
Approximate number of seeds per gram (number)
Approximate number of seed per ounce (number) 21
Yes
Elymus macrourus 
Tufted Wheatgrass 8 83
260 – 322
(291)
(8644)
Elymus wawawaiensis
Snake River Wheatgrass 9 84
(305)
(8556)
Pure Seed Unit (PSU) #21 definition of AOSA (Anonymous. 2014c).

25. Discussion
The proposed new rules for ELMA7 and ELWA2 as resubmitted for review are “reasonable” and similar to other Elymus species currently published within AOSA Rules for Testing Seeds (Table 6A; Anonymous, 2014b).
ELMA7: 20C, first count 7 d, final count 21 d
ELWA2: 20C, first count 7 d, final count 14 d
The calculated working weights are similar to other Elymus species published within ASOA Rules for Seeds Testing (Table 2A; Anonymous. 2014b) and the Federal Seed Act Regulations (Anonymous, 2001).
ELMA7 had a Purity Analysis weight = 8 g, Noxious-weed seed exam = 84 g, approximate number of seed per gram = 291, and approximate seed per ounce = seeds.
ELWA2 replicated weights were used to calculate a Purity Analysis weight = 9 g, Noxious-weed seed exam = 84 g, approximate number of seed per gram = 305, and approximate seed per ounce = 8556 seeds.

26. Conclusions
The addition of Elymus macrourus (ELMA7, Northern wheatgrass) and Elymus wawawaiensis (ELWA2, Snake River wheatgrass) germination standards to the AOSA Rules for Testing Seeds will establish uniformity among native grass seed laboratories.
If the genus Elymus elymoides ssp. brevifolis “SSP. C” is reevaluated and changes in taxonomic nomenclature and results in it no longer being a subspecies, it could later be included within the protocol.

27. References Cited
Anonymous Federal Seed Act. Code of Federal Regulations. Title 7 – Agriculture. Volume: 3. Date: Original Date: Title: SUBCHAPTER K - FEDERAL SEED ACT Context: Title 7 - Agriculture. Subtitle B - Regulations of the Department of Agriculture. CHAPTER I - AGRICULTURAL MARKETING SERVICE, DEPARTMENT OF AGRICULTURE. SUBCHAPTER K—FEDERAL SEED ACT. PART 201—FEDERAL SEED ACT REGULATIONS, Weight of working sample subchapK.xml#seqnum (accessed 25 September 2015).
Anonymous. 2014a. AOSA. [CD] Association of Official Seed Analysts, Inc. AOSA Rules for Testing Seeds Volume 1. Table 2. Principles and Procedures AOSA, nd Avenue, Suite #1, Moline, IL.
Anonymous. 2014b. AOSA. [CD] Association of Official Seed Analysts, Inc. AOSA Rules for Testing Seeds Volume 1. Principles and Procedures AOSA, nd Avenue, Suite #1, Moline, IL.
Anonymous. 2014c. AOSA. [CD] Association of Official Seed Analysts, Inc. AOSA Rules for Testing Seeds Volume 1. Table 3A. Principles and Procedures. AOSA, nd Avenue, Suite #1, Moline, IL.

28.
Acknowledgements
Committee Members: Dr. Richard T. Koenig, Chair; Vicki Bradley, M.S; Dr. William J. Johnston