This study evaluated the effects of various cytokines on senescence in Venus flytraps (Dionaea muscipula) and African violets. The researchers tested four treatment groups: a positive control with the artificial cytokinin BAP, a negative control without cytokinin, and two groups using coconut water and coconut milk. They found high rates of contamination across the Venus flytrap samples, but less in the African violet plates. While results varied between trials, the study aims to modify methods to prevent contamination and analyze telomere lengths to understand the impact of cytokines on plant senescence.

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

2. Introduction
● Primary Objective
● Specific Goals of Research:
o Evaluate the health claims of coconut products
o Determine the impact of cytokinins on plant senescence
o Analyze telomere lengths of tissue samples from various
treatment groups

3. Background
● Coconut Water Product Popularity and Health Claims
o 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

4. Background
● Cytokinins and Plant Leaf Senescence
o Kinetin Discovery, 1955
o 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

5. Background
● Telomere Biology
o Protection Endcaps of
Chromosomes
▪ DNA Damage
▪ Losses from DNA
Replication
o Telomerase

6. Background
● Plant Tissue Culturing (Mineo 1990)
o Genetically Identical
o Callus Formation
▪ Parenchyma Cells
▪ Wound Response
▪ Temporary Plant ‘Stem Cell’

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

8. Methods: Tissue Preparation
● Media Mix
o Formula
● Plant Cuttings and Plating
o Venus Fly Trap
o African Violet

9. 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

10. Methods
Treatment Groups:
BAP= artificial cytokinin group
(Positive control)
NC= No cytokinin
(Negative control)
CW= Coconut Water
CM= 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.

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

12. 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.

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

14. 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

15. Methods: Tissue Analysis
● DNA Extraction and Purification
● PCR Amplification
● DNA Analysis
o Gel Electrophoresis
o Southern Blot Method and TeloTool
o Problems and Other Options

16. Results
● Contamination of Samples
o Comparison of Treatment Groups
o Extent of Contamination

17. 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)

18. 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.

19. 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.

20. Conclusions
● Significance of Positive Control
● Future Steps
o Modifications to Media Formula
o Prevention of Contamination
o Analysis of Telomere Lengths

21. Acknowledgements
Dr. Jean Doty
Division of Natural Sciences, UMF
Emilie Morocco

22. 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.

23. Questions?