1. Agar Media Corn and Tomato 2015 National Referee
AOSA/SCST
2016 Annual Meeting
Melissa Phillips
Monsanto
Seed Technology Center
Waterman, Illinois

2. Purpose
The purpose of the referee was to introduce Agar (plant culturing media) as a potential candidate for use in germination testing.

3. Participants 24 Labs in total signed up to participate
20 labs to test corn
13 Labs to test tomato
Data returned (August –September 2015)
14 Labs returned corn data
8 Labs returned tomato data

4. 2015 Protocol Corn and Tomato 3 samples labeled 1-3 & 4-6
Samples 1-3 tested using the in-house AOSA approved method and agar media
Process was to be repeated 5-7 days later with samples 4-6 for repeatability and establish in lab variability
Agar concentration to be used 0.6%-1%

5. Corn
2015 Referee
Corn:
The mean germination was lower for Agar. The repeatability and reproducibility were also not as good as the current methods.
The data plot illustrates the result of a few labs with extreme data.

6. (Similar values for current and new methods are ideal)
2015 Referee
Conclusion: Corn Repeatability (within the same lab)
Crop
Method
Percent Germination
fr2 Sr
Corn
Agar
92.54
1.71
34.32
Current
93.94
1.32
27.43
Statistics courtesy of Paul Cannon-Monsanto
Repeatability is the closeness of the agreement between the results of the same measure and carried out in the same conditions.
Sr- Standard Deviation (lower the number the less variability)
fr2 –Reproducibility (> 1 is a sign of more variability than expected)
(Similar values for current and new methods are ideal)

7. (Similar values for current and new methods are ideal)
2015 Referee
Conclusion: Corn Reproducibility (across multiple labs)
Crop
Method
Percent Germination
fr2 SR
Corn
Agar
92.54
0.92
2.52
Current
93.94
0.34
1.39
Statistics courtesy of Paul Cannon-Monsanto
Reproducibility is the precision under reproducibility conditions, i.e. conditions where test results are obtained with the same method on identical test items in different laboratories with different operators using different equipment.
SR- Standard Deviation (lower the number the less variability)
fr2 - Reproducibility across labs (> 1 is a sign of more variability than expected)
(Similar values for current and new methods are ideal)

8. Study Conclusion 9 Months later…..
Referee data was not supportive of a rule proposal at the time
Further research needed – especially regarding large rounds
Continue research and optimization over the summer
9 Months later…..

9. Corn Today
100 samples
Further studies showed that the concentration of agar plays a factor on germination especially round corn seed.
After several variations of methods and concentrations the best correlation between paper media (CCP) and agar was a higher concentration than previous thought.

10. Conclusion: Tomato Repeatability (within the same lab)
2015 Referee
Crop
Method
Percent Germination
fr2 Sr
Tomato
Agar
89.60
1.79
40.88
Current
89.86
1.29
33.79
Statistics courtesy of Paul Cannon - Monsanto
Repeatability is the closeness of the agreement between the results of the same measure and carried out in the same conditions.
Sr- Standard Deviation (lower the number the less variability)
fr2 –Reproducibility within a lab (> 1 is a sign of more variability than expected)
(Similar values for current and new methods are ideal)

11. Tomato 2015 Referee

12. Conclusion: Tomato Reproducibility (across multiple labs)
2015 Referee
Crop
Method
Percent Germination
fr2 SR
Tomato
Agar
89.60
0.24
1.50
Current
89.86
0.48
2.09
Statistics courtesy of Paul Cannon-Monsanto
The reproducibility is the precision under reproducibility conditions, i.e. conditions where test results are obtained with the same method on identical test items in different laboratories with different operators using different equipment.
fR2 –Reproducibility (> 1 is a sign of more variability than expected)
SR- The average of the variance of results across all labs (similar number to the other method is desired.)
The mean germination, repeatability, and reproducibility are all comparable for the agar and current methods.

13. Tomato Referee 2016 5 labs 6 samples Agar vs. Current Method
Conclusion: Tomato on agar has consistently shown equivalency when compared to the current approved media (top of paper & rolled towel) in 2 large referees and 1 small over 2 years and many laboratories.

14. Moving Forward Proposals 12 & 13 Seed Issues Forum
Agar as a media & Tomato on agar
Seed Issues Forum
Live Seedlings!
Sign up for future referees

15. Special Thanks AgReliant Genetics Dow Agrosciences Monsanto Company
for their support by donating seed for the project

16. Special Thanks to Participants
AgReliant Agri Seed Testing* Alforex Seeds California Dept Food & Ag California Seed & Plant Lab Dow Agrosciences Eurofins BDI Florida Dept of Ag Georgia Dept Ag Growmark H M Clause Illinois Crop Improvement Incotec Indiana Crop Improvement Kent Agri Lab Michigan Crop Improvement Monsanto NST Labs Ohio Seed Improvement Sakata Seed Sakata Seed PNW SGS Syngenta Syngenta Veg USDA Forestry* USDA- SRTD USDA/ ARS Plant Intro
*Seedlings for meetings

17. References
Miles, S.R. (1963), Handbook of tolerances and measures of precision for seed testing. Proc. Int. Seed Test. Ass. 28 (3).
Nijënstein, J.H. (2013). Revised method and ISTA method validation report for the germination of Lolium x bouceanum, L. multiflorum, L. perenne, Festuca filiformis, F. heterophylla, F. ovina, F.rubra, Poa nemoralis, P. palustris, P. pratensis. ISTA Method Validation Report. Method Validation Reports on Rules Proposals for the International Rules for Seed Testing 2014 Edition, 2013, 2-33.