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EFFECTS OF VARIOUS CYTOKINES ON SENESCENCE IN VENUS FLYTRAPS (DIONAEA MUSCIPULA)Jordan Horrocks and Nickolas Bray
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Introduction● Primary Objective
● Specific Goals of Research: o Evaluate the health claims of coconut productso Determine the impact of cytokinins on plant
senescenceo Analyze telomere lengths of tissue samples from
various treatment groups
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Background ● Coconut Water Product Popularity and Health
Claimso Anti-aging Properties (Carlson 2011)o IV Replacement (Campbell-Falck et al 2000)o Anti-inflammatory (Ajeigbe et al 2011)
● Coconut Water vs. Coconut Milk
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Background● Cytokinins and Plant Leaf Senescence
o Kinetin Discovery, 1955o Kinetin “retards senescence in plants, [and] delays
aging in human cells in culture” (Barciszewski et al 2006).
o Cytokinin’s Effects on ▪ Cell Growth and Development▪ Plant Leaf Senescence
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Background● Telomere Biology
o Protection Endcaps of Chromosomes▪ DNA Damage▪ Losses from DNA
Replicationo Telomerase
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Background● Plant Tissue Culturing (Mineo 1990)
o Genetically Identicalo Callus Formation
▪ Parenchyma Cells▪ Wound Response▪ Temporary Plant ‘Stem Cell’
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Model Organism Evolution● Tomatoes● Tobacco● Venus Fly Traps● African Violet
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Methods: Tissue Preparation● Media Mix
o Formula● Plant Cuttings and Plating
o Venus Fly Trapo African Violet
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Methods: Growth Media● Basic formula for callus generation (for
250 mL)o DI Water: 239.7 mL
o Thickening Agent: Agar- 2g
o Carbon Source: Sucrose- 7.5 g
o Nutrient Mixture: MS Basal Salt mixture- 0.55 g
o Cytokinin (optional): 0.25 mL
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MethodsTreatment Groups:BAP= artificial cytokinin group (Positive control)NC= No cytokinin
(Negative control)CW= Coconut WaterCM= Coconut Milk
Callus Media was made up for each of the four treatment groups, and left to harden over the weekend. None displayed contamination prior to plant plating.
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Methods
Plant specimens were obtained, and pith segments were isolated to be plated.
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Methods
Each pith segment was soaked in 90% Ethanol for 15 seconds, followed by 15 seconds in DI water, before being cut into 5mm segments. These segments were then placed into one of the 40 media plates.
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Methods
Once all 40 plates contained a plant segment, they were sealed with Parafilm, to prevent both desiccation and contamination.
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Method Modifications● Venus Flytrap plates
showed contamination in over ⅔ of samples by the end of week one:
▪ Repeat trial, with modifications
▪ New model organism: African Violet- Thicker Stem, easier to extract pith
▪ 75% isopropanol▪ New, sterile clean room
environment
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Methods: Tissue Analysis● DNA Extraction and Purification● PCR Amplification● DNA Analysis
o Gel Electrophoresiso Southern Blot Method and TeloToolo Problems and Other Options
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Results● Contamination of Samples
o Comparison of Treatment Groupso Extent of Contamination
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Results
These charts show the different distribution of non-contaminated samples over time across treatment groups. There is variation between the two trials, but we can only speculate the cause.
CM (8) CW (6) BAP (6) NC (3)
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Results
These photos show the major types of contamination found in Venus Flytrap plates. As seen, the thin nature of the plants’ tissue caused them to blacken within the first week, causing the first type of contamination to be far more typical.
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Results
These photos show some of the variety found in African Violet plates. As seen, the plants’ tissue is much thicker, causing it to maintain its color. Curiously, multiple types of contamination within individual samples was far more common, as well as a wider variety of bacteria.
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Conclusions● Significance of Positive Control● Future Steps
o Modifications to Media Formulao Prevention of Contaminationo Analysis of Telomere Lengths
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Acknowledgements Dr. Jean Doty
Division of Natural Sciences, UMF
Emilie Morocco
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Citations• Ajeigbe KO, Ndaman ZA, et al. Anti-nociceptive and anti-inflammatory potential of
coconut water (Cocos Nucifera L.) in rats and mice. Aust. J. Basic & Appl. Sci. 2011;5(9);1116-1122.
• Barciszewski J. Kinetin-- a multiactive molecle. International Journal of Biological Macromlecles. 2007;40(3);182-192.
• Campbell-Falck D, Thomas T, et al. The intravenous use of coconut water. American Journal of Emergency Medicine. 2000; 18(1);108-111.
• Mineo L. Plant tissue culture techniques. Tested Studies For Laboratory Teaching. 1990;11(9);151-174.
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Questions?