Presentation on theme: "Can Melatonin Break Seed Dormancy? Algerr Remy John Eakin Mentor: Dr. Magaly Rincόn-Zachary."— Presentation transcript:
Can Melatonin Break Seed Dormancy? Algerr Remy John Eakin Mentor: Dr. Magaly Rincόn-Zachary
Purpose: To investigate if melatonin substitutes for the stratification and photodormancy requirements in several plant seed species
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.
What is Photodormancy? Photodormancy is a condition in which seeds of some plant species require light in order to germinate.
Preliminary Testing Preliminary experiments were preformed to confirm germination rates, dormancy status, and germination time of each species.
Experimental Design 3 species were tested: Triticum aestivum Wheat Arabidopsis thaliana Thale Cress Hypericum perforatum St. Johns Wort
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
Experiment 1 Possible Problems Did the seeds used still require stratification?
Does Melatonin Substitute for Light to Break Seed Photodormancy? Part 1 Used different melatonin concentrations Sterilized seeds to limit contamination Determined germination rates
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).
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 …
Preparing the Seeds: Sterilization – 5 min in a 5% Bleach, 0.1% SDS (Sodium Dodecyl Sulfate) mixture. – Rinsing with distilled H 2 0
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 mM phosphate buffer (pH 5.83) was added to control dishes
Observing seeds for signs of germination: Seeds were observed under a dissection microscope Germination rates were tabulated in Excel
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?
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
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?
Was the Phosphate Buffer Responsible for Breaking Seed Dormancy? Taraxacum officinale & Nicotiana benthamiana 4 treatments: Light H 2 O Dark H 2 O Light phosphate buffer Dark phosphate buffer Arabidopsis thaliana 4 treatments: 4 °C phosphate buffer 4 °C H 2 O 27 °C phosphate buffer 27 °C H 2 O
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 seeds not require stratification?
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
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?
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(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, 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.