National Referee Agar Media Corn Germination 2016 Melissa Phillips Monsanto Seed Technology Center Waterman, Illinois USA

Purpose The purpose of the referee was to introduce Agar (plant culturing media) as a potential candidate for use in germination testing for corn. Participants 16 Labs in total signed up to participate Agar was provided upon request to 7 labs Seed was sent out June 2016 Data was returned August- Sept 10 labs returned data

Previous Studies Protocol Kept with more traditional studies of “open protocols” Agar requires more specific requirements 6 corn samples Samples tested using in-house AOSA approved method and agar media Process was to be repeated 5-7 days later to establish lab repeatability Agar concentration to be used 0.8%-1%

Agar results are on par with current methods with in each lab. Repeatability is the closeness of the agreement between the results of the same measure and carried out in the same conditions. 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. Variance Method Mean Germ Median Germ Repeatability* Reproducibility* Part to Part Interaction Total Agar 84.3 88 28.9 (17%) 29.0 (17%) 99.2 (58%) 15.1 (9%) 172.2 Current 87.1 90 24.2 (19%) 4.3 (3%) 89.0 (70%) 10.5 (8%) 128.0 *Adequate measurement systems have total Repeatability + Reproducibility less than 10% of total variation Agar results are on par with current methods with in each lab. Agar results are less reproducible than current methods when compared across multiple labs. Statistics supplied by Paul Cannon and Kirk Remund- Monsanto

Corn Germination Reproducibility

Significant Outliers; Why? Some labs were in alignment, others were not Post referee survey Current Method CCP or CCP/Sand vs. Rolled Towel Experience with Agar First Time to Experienced Equipment used for agar preparation Stove Microwave Hotplate Autoclave Significant Outliers; Why? What is Occurring?

Corn Germination based on agar experience No conclusions can be made based on experience * sample numbers are not large enough to be significant

Corn germination based on media * sample numbers are not large enough to be significant

Conclusion Study results were not supportive of a 2016 rule proposal for corn germination Results show that there is still reproducibility issues across some labs In-lab repeatability is on par with current approved methods The use of specific protocols increases the repeatability of the agar method

Moving Forward Thank you! Further analysis of factors impacting repeatability and reproducibility Future Targeted/ Strategic Study Protocol of corn agar that will allow us to control critical to quality control variables Thank you! Special thanks to all of the referee participants!