Can Melatonin Break Seed Dormancy?

1. Can Melatonin Break SeedDormancy? Algerr Remy John Eakin Mentor: Dr. Magaly Rincόn-Zachary

2. Purpose: To investigate if melatonin substitutes for the stratification and photodormancyrequirements in several plant seed species

3. What is Stratification? Stratification is a treatment given to seeds of some plant species that require a period of cold temperatures (4 °C- 8 °C) in order to germinate.

4. What is Photodormancy? Photodormancy is a condition in which seeds of some plant species require light in order to germinate.

5. Preliminary Testing Preliminary experiments were preformed to confirm germination rates, dormancy status, and germination time of each species.

6. Stratification Experiments

7. Experimental Design 3 species were tested: Triticum aestivum Wheat • Arabidopsis thaliana Thale Cress • Hypericum perforatum St. John’s Wort

8. Does Melatonin Substitute for Stratification to Break Seed Dormancy? Treatments: • Treatment 1: 1 nM Melatonin at 27 °C • Treatment 2: 0 Melatonin at 27 °C • Treatment 3: 0 Melatonin at 4 °C for 24 hours => 27 °C

11. Experiment 1 Possible Problems • Did the seeds used still require stratification?

12. PhotodormancyExperiments

13. Experimental Design 9 species were tested: • Barbareaverna • Upland cress • Centaureacyanus • Bachelor’s blue button • Coreopsis grandiflora • Largeflower tickseed • Echinacea purpurea • Purple coneflower • Nicotianabenthamiana • Lepidumsativum • Curled pepper cress • Nicotianatabacum • Cultivated tobacco • Taraxacumofficinale • Dandelion • Rudbeckahirta • Black-eyed susan

14. Experimental Design Two test groups: In each treatment: 9 Petri dishes 20 seeds per dish • Light Treatment • Dark Treatment

15. Experimental Design • Seeds were observed under a dissection microscope at 15X magnification • Germination rates were tabulated in Excel

16. Results

17. Does Melatonin Substitute for Light to Break Seed Photodormancy?Part 1 • Used different melatonin concentrations • Sterilized seeds to limit contamination • Determined germination rates

18. Preparation: • 4 melatonin concentration treatment groups for each seed species, as well as an untreated control group. • 1 nM (10-9 M), 1 pM (10-12 M), 1 ƒM (10-15 M), and 1 aM (10-18 M).

19. Serial Dilutions: • A 0.1 M melatonin stock solution in 95% ethanol. • Treatment solutions prepared using a serial dilution method 0.1 M → 0.01 M → 0.001 M → 0.0001 M …

20. Preparing the Seeds: • Sterilization • 5 min in a 5% Bleach, 0.1% SDS (Sodium Dodecyl Sulfate) mixture. • Rinsing with distilled H20

21. Treatment: • Petri dishes were lined with filter paper • Filter paper was saturated with 2.5 mL of melatonin treatment + 2.5 mL of 1.0 mM phosphate buffer (pH 5.83) • 5 mL of 1.0 mMphosphate buffer (pH 5.83) was added to control dishes

22. Observing seeds for signs of germination: • Seeds were observed under a dissection microscope • Germination rates were tabulated in Excel

26. Possible Problems: • The control group had twice the concentration of phosphate buffer, did this effect the germination rate? • Did light exposure during seed sterilization break dormancy? • Did seeds lose viability during the sterilization process?

27. Does Melatonin Substitute for Light to Break Seed Photodormancy?Part 2 Experiment 3 • No sterilization • Equal proportion of buffer in all treatments • Untreated light controls were added

32. Possible Problems: • Was the phosphate buffer responsible for breaking seed dormancy? • Did the state of the aluminum foil lead to light exposure? • What happened to the pM treatment? • Was the concentration of melatonin too low?

33. Was the Phosphate Buffer Responsible for Breaking Seed Dormancy? • Taraxacumofficinale&Nicotianabenthamiana • 4 treatments: • Light H2O • Dark H2O • Light phosphate buffer • Dark phosphate buffer • Arabidopsis thaliana • 4 treatments: • 4 °C phosphate buffer • 4 °C H2O • 27 °C phosphate buffer • 27 °C H2O

37. Conclusions • The phosphate buffer does not appear to be the cause of high germination rates in the treatments. • Experiment needs to be replicated to ensure consistent results. • Photodormant plants are still photodormant. • Why were the control germination rates so high in previous experiments? • Do Arabidopsis seedsnot require stratification?

38. Continuing Research on Melatonin Overcoming Dormancy in Seeds • Repeat phosphate experiment: • Rule out phosphate as a dormancy breaker • Repeat Melatonin experiments: • Find potential errors from the previous experimental design • Can the experiments be performed without the buffer? • Can a different buffer be used? • Increased Melatonin concentrations • Stratification Experiments: • Find seeds that require stratification • Test times needed for stratification • Observe whether melatonin can break dormancy in seeds requiring stratification

39. Does Melatonin Speed Up the Life Cycle of Plants? Preliminary research suggests that Melatonin may speed up the life cycle of plants, an experiment must be designed to test this phenomenon. • Does melatonin shorten the life cycle? • How do different concentrations affect the life cycle? • What is the melatonin optimum concentration for the fastest life cycle? • Are the effects similar across several species of plants?

40. Literature Cited • Arnao, M.B. and J. Hernández-Ruiz. (2006). The Physiological Function of Melatonin in Plants. Plant Signaling and Behavior, 1(3), 85-95. • Finch-Savage, W.E. and G. Leubner-Metzger. (2006). Seed Germination and the Control of Dormancy. New Phytologist, 171, 501-523. doi:10.1111/j.1469-8137.2006.01787.x • Harden, A.L.(2013). Melatonin Accelerates Germination and Flowering and Alters Other Developmental Processes in Tobacco(NicotianatabacumCV. Havana). Unpublished Manuscript. • Kolář, J., Johnson, C.H. and IvanaMacháčková.(2003). Exogeneously Applied Melatonin (N-acetyl-5-methoxytryptamine). PhysiologiaPlantarum, 118, 601-612. • Paredes, S.D., Korkmaz, A., Manchester, L.C., et al. (2009). Phytomelatonin: A Review. Journal of Experimental Botany, 60(1), 57-69. doi:10.1093/jxb/ern284.