7 Experimental Design 3 species were tested: Arabidopsis thaliana Thale CressHypericum perforatumSt. John’s WortTriticum aestivumWheat
8 Does Melatonin Substitute for Stratification to Break Seed Dormancy? Treatments:Treatment 1: 1 nM Melatonin at 27 °CTreatment 2: 0 Melatonin at 27 °CTreatment 3: 0 Melatonin at 4 °C for 24 hours => 27 °C
17 Does Melatonin Substitute for Light to Break Seed Photodormancy? Part 1 Used different melatonin concentrationsSterilized seeds to limit contaminationDetermined 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: 0.1 M → 0.01 M → 0.001 M → 0.0001 M … A 0.1 M melatonin stock solution in 95% ethanol.Treatment solutions prepared using a serial dilution method0.1 M → 0.01 M → M → 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 mL of 1.0 mM phosphate buffer (pH 5.83)5 mL of 1.0 mM phosphate buffer (pH 5.83) was added to control dishes
22 Observing seeds for signs of germination: Seeds were observed under a dissection microscopeGermination 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 3No sterilizationEqual proportion of buffer in all treatmentsUntreated 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? Taraxacum officinale & Nicotiana benthamiana4 treatments:Light H2ODark H2OLight phosphate bufferDark phosphate bufferArabidopsis thaliana4 treatments:4 °C phosphate buffer4 °C H2O27 °C phosphate buffer27 °C H2O
37 ConclusionsThe 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 seeds not require stratification?
38 Continuing Research on Melatonin Overcoming Dormancy in Seeds Repeat phosphate experiment:Rule out phosphate as a dormancy breakerRepeat Melatonin experiments:Find potential errors from the previous experimental designCan the experiments be performed without the buffer?Can a different buffer be used?Increased Melatonin concentrationsStratification Experiments:Find seeds that require stratificationTest times needed for stratificationObserve 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 CitedArnao, M.B. and J. Hernández-Ruiz. (2006). The Physiological Function of Melatonin in Plants. Plant Signaling and Behavior, 1(3),Finch-Savage, W.E. and G. Leubner-Metzger. (2006). Seed Germination and the Control of Dormancy. New Phytologist, 171, doi: /j xHarden, A.L.(2013). Melatonin Accelerates Germination and Flowering and Alters Other Developmental Processes in Tobacco(Nicotiana tabacum CV. Havana). Unpublished Manuscript.Kolář, J., Johnson, C.H. and Ivana Macháčková.(2003). Exogeneously Applied Melatonin (N-acetyl-5-methoxytryptamine). Physiologia Plantarum, 118,Paredes, S.D., Korkmaz, A., Manchester, L.C., et al. (2009). Phytomelatonin: A Review. Journal of Experimental Botany, 60(1), doi: /jxb/ern284.