can melatonin break seed dormancy?
DESCRIPTION
Can Melatonin Break Seed Dormancy?. Algerr Remy John Eakin Mentor: Dr. Magaly Rinc ό n-Zachary. Purpose:. To investigate if melatonin substitutes for the stratification and photodormancy requirements in several plant seed species. What is Stratification?. - PowerPoint PPT PresentationTRANSCRIPT
Can Melatonin Break Seed Dormancy?
Algerr Remy John EakinMentor: Dr. Magaly Rincόn-Zachary
Purpose:To investigate if melatonin substitutes for thestratification and photodormancy requirements inseveral plant seed species
What is Stratification?
Stratification is a treatment given to seeds ofsome plant species that require a period of coldtemperatures (4 °C- 8 °C) in order togerminate.
What is Photodormancy?
Photodormancy is a condition in which seeds ofsome plant species require light in order togerminate.
Preliminary Testing
Preliminary experiments were preformed toconfirm germination rates, dormancy status, and germination time of each species.
Stratification Experiments
Experimental Design
3 species were tested:
• Triticum aestivum Wheat
• Arabidopsis thaliana Thale Cress
• Hypericum perforatum St. John’s 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
0 M Mel 1 nM Mel Cold Stratified0%
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Effect of Melatonin on Wheat Seed Germination
Treatment
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O
0 M Mel 1nM Mel Cold Stratified0%
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Effect of Melatonin on Arabidopsis thaliana Seed Germination
Treatment
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0 1 nM Mel
Experiment 1 Possible Problems
• Did the seeds used still require stratification?
PhotodormancyExperiments
Experimental Design9 species were tested:
• Lepidum sativum Curled pepper cress
• Nicotiana tabacum Cultivated tobacco
• Taraxacum officinale Dandelion
• Rudbecka hirta Black-eyed susan
• Barbarea verna Upland cress
• Centaurea cyanus Bachelor’s blue button
• Coreopsis grandiflora Largeflower tickseed
• Echinacea purpurea Purple coneflower
• Nicotiana benthamiana
Experimental Design
Two test groups:• Light Treatment• Dark Treatment
In each treatment:• 9 Petri dishes • 20 seeds per dish
Experimental Design
• Seeds were observed under a dissection microscope at 15X magnification
• Germination rates were tabulated in Excel
Results
Photodormant Non-Photodormant
Taraxacum officinale Coreopsis grandiflora
Nicotiana benthamiana Rudbecka hirta
Nicotiana tabacum Lepidum sativum
Barbarea verna
Centaurea cyanus
Echinacea purpurea
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 H20
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
0%
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Perc
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1 nM1 pM1 fM1 aM0
Melatonin Concentration
Effect of Melatonin on Nicotiana benthamaina Seed Germination in the Dark
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Effect of Melatonin on Nicotiana tabacum Seed Germination in the Dark
Melatonin Concentration
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0 1 aM 1 ƒM 1 pM 1 nM
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Effect of Melatonin on Taraxacum officinale Seed Germination
in the Dark
Melatonin Concentration
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0 1 aM 1 ƒM 1 pM 1 nM
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
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Effect of Melatonin on Nicotiana benthamiana Seed Germina-
tion in the Dark
Melatonin Concentration
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1 aM 1 ƒM 1 pM 1 nM0
0%
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Effect of Melatonin on Nicotiana tabacum Seed Germination in
the Dark
Melatonin Concentration
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0 1 aM 1 ƒM 1 pM 1 nM Light Control 0M
0%
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Effect of Melatonin on Taraxacum officinale Seed Germination
in the Dark
Melatonin Concentration
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0 1 aM 1 ƒM 1 pM 1 nM UntreatedLight Control
0 M
0%
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Effect of Melatonin on Taraxacum officinale Seed Germination
in the Dark
Melatonin Concentration
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0 1 aM 1 ƒM 1 pM 1 nM UntreatedLight Control
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 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
0%
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Effect of Phosphate Buffer on Taraxacum officinale Germination
Treatments
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Dark H2O Dark Phosphate
Light H2O LightPhosphate
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Effect of Phosphate Buffer on Nicotiana benthamiana Germination
Treatment
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H2O Dark Buffer Dark H2O Light Buffer Light
0%
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2%
8%
17%
23%
Effect of the Phosphate Buffer on Arabidopsis thaliana Seed Germina-
tion
Treatment
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H2O 4 °C Buffer 4 °C H2O 27 °C Buffer 27 °C
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.