1. Agar Media Corn and Tomato National Referee 2014
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 25 Labs in total signed up to participate
19 labs to test Corn
19 Labs to test Tomato
Agar was provided upon request to 14 labs
Data returned (December 2014-April 2015)
12 Labs returned Corn data
11 Labs returned Tomato data
One data set was thrown out as replicates did not add to 100.

4. 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. Overall Comments Easier than I thought it would be.
Easy view of roots
Analyst felt it was easier to remove seedlings
Allows us to use various sized containers
Moisture- Like other medias will dry out if left open
Uncovered trays dried out
Majority of comments
Agar preparation
Agar never fully solidified
Different texture between batches
Agar concentrations used
Agar preparation- Need to make temperature
With any new media or procedure; time and experience is needed to fully understand and optimize conditions in your own lab.

6. Corn
Corn:
The mean germination was significantly 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.

7. (Similar values for current and new methods are ideal)
Conclusion: Corn Repeatability (Within the same lab)
Crop
Method
Percent Germination
fr2 Sr
Corn
Agar
90.78
2.27
0.61
Current
94.87
0.78
0.13
Statistics courtesy of Tim Perez- 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)

8. (Similar values for current and new methods are ideal)
Conclusion: Corn Reproducibility (across multiple labs)
Crop
Method
Percent Germination
fr2 SR
Corn
Agar
90.78
5.33
5.62
Current
94.87
0.30
0.19
Statistics courtesy of Tim Perez- 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)

9. In Lab Existing Agar Method
An Example Lab
Corn
Lab
AOSA
Method
(Towel 25°c)
Referee
Protocol
(0.7% conc.)
In Lab Existing Agar Method
Sample 1 92 56 91
Sample 3 94 63
Sample 5 98 35 97
Protocol stated to use approximate volumes depending on tray type for a target of about a centimeter of agar on the tray.
This lab’s standard agar depth is 2 inches.

10. 3 Outlier’s removed Repeatability- Within Lab
Crop
Method
Percent Germination
fr^2 Sr
Corn
Agar
94.12
0.17
0.98
Current
94.87
0.13
0.78
Reproducibility- Across Labs
Crop
Method
Percent Germination
fr^2 SR
Corn
Agar
94.12
0.51
0.36
Current
94.87
0.30
0.19
9 labs remaining
Statistics courtesy of Tim Perez- Monsanto
Corn: When the method is optimized in each lab; the mean germination, repeatability, and reproducibility are all comparable for the Agar and current methods.

11. Tomato

12. Conclusion: Tomato Repeatability (Within the same lab)
Crop
Method
Percent Germination
fr2 Sr
Tomato
Agar
92.66
0.19
1.14
Current
92.72
0.22
1.21
Statistics courtesy of Tim Perez- 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)

13. Conclusion: Tomato Reproducibility (across multiple labs)
Crop
Method
Percent Germination
fr2 SR
Tomato
Agar
92.66
0.84
0.99
Current
92.72
0.66
0.78
Statistics courtesy of Tim Perez- 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 (> 1 is a sign of more variability than expected)
(Similar values for current and new methods are ideal)
Tomato:
The mean germination, repeatability, and reproducibility are all comparable for the Agar and current methods.

14. Moving Forward Repeat the referee: Corn and Tomato
Immediately after the meeting
Sign Up at Seed Issues Forum
Participants – New and Old
Clarify the protocol
Include depth recommendations
Container requirements “to limit evaporation”

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

16. 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.