Agar Media Corn and Tomato National Referee 2014 Melissa Phillips Monsanto Seed Technology Center Waterman, Illinois

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

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.

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%

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.

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.

(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)

(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)

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.

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.

Tomato

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)

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.

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”

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

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.