Caffeine’s Effect on Mung Bean Germination and growth Todd Oravitz 9th grade central catholic

INSPIRATION

caffeine Naturally occurring substance Bitter, white purine compound Similar chemical structure to adenine and guanine Adenine and guanine found in DNA and bind to thymine and cytosine, respectively.

Caffeine effects Blocks adenosine receptors, leading to calcium loss in plant cells Low calcium can cause problems with Cell membrane permeability Cell plate formation

Caffeine effects Interferes with plant cytokinesis Stops Golgi vesicles from fusing with membranes by decreasing ATP activity Has been shown to inhibit cell division in plants

Caffeine in nature Pesticide-like protection to plants containing it Germination of competing seedlings may be slowed by plants depositing caffeine in nearby soil

Guarana plant Effective natural stimulant Seeds contain about twice the caffeine concentration as those from coffee

Guarana plant Naturally alters one’s perception of fullness, leading to weight loss FDA recognizes it as “generally safe”

Purpose To determine if caffeine has an effect on germination and growth of mung beans

HYPOTHESeS Null Caffeine will not have a significant effect on mung bean germination and growth Alternative Caffeine will have a significant effect on mung bean germination and growth

materials Seed starter trays Potting soil Mung beans Guarana – caffeine source Sunlight via window Room lights Tap water Pyrex 500mL measuring cup (to make test solution) 10mL measuring cup (for watering) Ruler Scientific scale (no continuous, dedicated light source)

procedure Planted mung beans 72 plants each in test and control groups 5 mL caffeine solution [200mg/L] given every other day to test group 5 mL tap water given every other day to control group Per research, 3.8cm is optimal depth for planting mung beans.

procedure Main shoot height of mung beans measured daily for 28 days Mung bean mass measured on day 28: Plant removed, rinsed with tap water and cut at ground level Above and below ground wet masses measured, then added for total Procedure repeated after air drying for three hours to obtain dry mass

Caffeine control DAY 28

Germination analysis No growth Growth Total Caffeine 51 21 72 Control 19 53 70 74 144 Highly significant difference in the NUMBER of mung beans that grew between caffeine and control groups. Χ2 = 28.466, p < 0.00001

conclusions Null hypothesis rejected Alternative hypothesis accepted – caffeine had a significant effect on mung bean germination and growth Specifically, it significantly decreased the number of mung beans that germinated

Question When caffeine group mung beans did germinate, did they exhibit similar growth characteristics to control?

CAFFEINE EFFECT ON SHOOT HEIGHT general linear modeling, p = 0.812 avg shoot height (mm) blue-caffeine green-control General Linear Modeling – way to evaluate how similar the 2 curves are; kind of like comparing the slope of 2 lines. Average growth per plant for plants that grew (eliminated those with no growth). day

Height analysis Daily average mung bean shoot height compared Only plants that germinated No significant difference between caffeine and control average daily shoot heights General Linear Modeling w/p value 0.812 – the 2 curves are NOT SIGNIFICANTLY DIFFERENT

AVG MASS/PLANT – WET mass (g) wet mass above ground below ground total T-test with significant p value only for below ground wet mass. Average wet mass/plant for plants that had growth (dropped ones that did not grow). wet mass above ground below ground total caffeine, g 0.297 0.261 0.558 control, g 0.259 0.308 0.567 p value 0.052 0.035 0.785 significant? no yes

AVG MASS/PLANT – DRY dry mass above ground below ground total T-test with significant p value only for above ground dry mass. Average dry mass/plant for plants that grew (eliminated ones that did not grow). dry mass above ground below ground total caffeine, g 0.224 0.129 0.353 control, g 0.176 0.142 0.318 p value 0.009 0.262 0.125 significant? yes no

ABOVE/BELOW GROUND WET MASS RATIO

Mass analysis T-test done for all 6 subgroups Significant difference seen in 2 Below wet (p=0.035) and above dry (p=0.009) No significant difference in the other 4 Above wet, total wet, below dry and total dry Overall, then, no significant difference between caffeine and control mass, when broken down into the subgroups. T-tests done for all 6 subgroups.

HEIGHT, WET MASS CORRELATION blue – caffeine; R=0.963 green – control; R=0.807 p<0.001 height (mm) Only plotted height vs mass for those plants that grew (dropped ones with no growth). total wet mass (g)

HEIGHT, DRY MASS CORRELATION blue – caffeine; R=0.941 green – control; R=0.815 p<0.001 height (mm) Only plotted height vs mass for those plants that grew (dropped ones with no growth). total dry mass (g)

Height vs mass analysis Height vs total wet and dry mass Only plants that germinated Pearson correlation coefficient Height correlated with mass in both wet and dry groups PCC – way to look at how shoot height correlated with mass. NO SIGNIFICANT DIFFERENCES BETWEEN HEIGHT/MASS OR CAFFEINE/CONTROL. More evidence that when mung beans grew in the caffeine group, they grew just like those in the control group.

conclusions Null hypothesis rejected Alternative hypothesis accepted – caffeine had a significant effect on mung bean germination and growth Specifically, it reduced the number of plants that germinated

conclusions Mung beans in the caffeine group that did germinate, however, showed similar growth to control No significant differences in Average daily shoot height Average total wet mass Average total dry mass Kind of a secondary conclusion – when caffeine mung beans grew they were the same as control plants.

Limitations and extensions Did not control soil content Short drying time Inconsistent lighting Extensions Different caffeine concentrations Correlate pre-planting mung bean mass with germination Defined non-sunlight source

bibliography ag.arizona.edu/pubs/garden/mg/soils/types.html Arnaud, M.J. 1987. The pharmacology of caffeine. Prog. Drug Res. 31: 273-313. Bonsignore, C.L, and Hepler, P.K. “Caffeine Inhibition of Cytokinesis: Dynamics of Cell Plate Formation- Deformation in vivo.” Protoplasma. 129, 28-35; 1985. en.wikipedia.org/wiki/guarana Etherdon, G.M., and M.S. Kochar. 1993. Coffee: Facts and controversies. Arch. Fam. Med. 2(3):317-322. extension.oregonstate.edu/lane/sites/default/files/docume nts/cffee07.pdf Hazardous Substances Data Bank. 1997. Caffeine. HSDB number 36. Bethesda, MD: National Library of Medicine.

Bibliography Hepler, P.K. “Calcium: A Central Regulator of Plant Growth and Development.” Plant Cell 2005; 17; 2142-55. Kabagambe, Edmond K. "Benefits and Risks of Caffeine and Caffeinated Beverages." UpToDate. Wolters Kluwer Health, 27 Feb 2013. Lopez-Saez, J.F. et al. “ATP level and caffeine efficiency on cytokinesis inhibition in plants.” Eur J Cell Biol. 1982 Jun; 27(2): 185-90. Nathanson, J.A. “Caffeine and related methylxanthines: possible naturally occurring pesticides.” Science. 226 (4671), 184-7; 1984. www.hort.purdue/edu/ext/groundsforgardening.html www.hort.purdue.edu/newcrop/afcm/mungbean.html

Acknowledgements Thanks to Mr. Krotec for support and guidance throughout the experiment. Thanks to James Ibinson, MD, PhD, for help with statistical analysis. Thanks to my parents for helping me with ideas and suggestions, as well as supply of materials.

ANOVA TESTING – above/below wet mass Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column 1 21 6.233 0.29681 0.004239 Column 2 5.489 0.261381 0.006237 Column 3 53 13.718 0.25883 0.008701 Column 4 16.345 0.308396 0.007563 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 0.078898 3 0.026299 3.588881 0.015321 2.667443 Within Groups 1.055238 144 0.007328 Total 1.134137 147  

ANOVA TESTING – above/below dry mass Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column 1 21 4.701 0.223857 0.004027 Column 2 2.71 0.129048 0.001461 Column 3 53 9.332 0.176075 0.005073 Column 4 7.547 0.142396 0.002345 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 0.133358 3 0.044453 12.91884 1.6E-07 2.667443 Within Groups 0.495492 144 0.003441 Total 0.62885 147  

ABOVE/BELOW GROUND dry MASS RATIO