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

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0 M Mel 1nM Mel Cold Stratified0%

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Effect of Melatonin on Arabidopsis thaliana Seed Germination

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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

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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

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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

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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

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Effect of Melatonin on Taraxacum officinale Seed Germination

in the Dark

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0 1 aM 1 ƒM 1 pM 1 nM UntreatedLight Control

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Effect of Melatonin on Taraxacum officinale Seed Germination

in the Dark

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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

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Effect of Phosphate Buffer on Taraxacum officinale Germination

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Dark H2O Dark Phosphate

Light H2O LightPhosphate

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Effect of Phosphate Buffer on Nicotiana benthamiana Germination

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H2O Dark Buffer Dark H2O Light Buffer Light

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Effect of the Phosphate Buffer on Arabidopsis thaliana Seed Germina-

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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.