EFFECTS OF VARIOUS CYTOKINES ON SENESCENCE IN VENUS FLYTRAPS (DIONAEA MUSCIPULA)Jordan Horrocks and Nickolas Bray

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

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

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

Background● Telomere Biology

o Protection Endcaps of Chromosomes▪ DNA Damage▪ Losses from DNA

Replicationo Telomerase

Background● Plant Tissue Culturing (Mineo 1990)

o Genetically Identicalo Callus Formation

▪ Parenchyma Cells▪ Wound Response▪ Temporary Plant ‘Stem Cell’

Model Organism Evolution● Tomatoes● Tobacco● Venus Fly Traps● African Violet

Methods: Tissue Preparation● Media Mix

o Formula● Plant Cuttings and Plating

o Venus Fly Trapo African Violet

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

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.

Methods

Plant specimens were obtained, and pith segments were isolated to be plated.

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.

Methods

Once all 40 plates contained a plant segment, they were sealed with Parafilm, to prevent both desiccation and contamination.

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

Methods: Tissue Analysis● DNA Extraction and Purification● PCR Amplification● DNA Analysis

o Gel Electrophoresiso Southern Blot Method and TeloToolo Problems and Other Options

Results● Contamination of Samples

o Comparison of Treatment Groupso Extent of Contamination

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)

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.

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.

Conclusions● Significance of Positive Control● Future Steps

o Modifications to Media Formulao Prevention of Contaminationo Analysis of Telomere Lengths

Acknowledgements Dr. Jean Doty

Division of Natural Sciences, UMF

Emilie Morocco

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.

Questions?