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It is common to joke among friends that consuming coffee can stunt a persons growth. The researchers tested this common theory in pea pollen tubes. Therein they observed that the introduction of caffeine did have a negative effect on pollen growth elongation in their pea samples. It was shown that the caffeine augmented antibodies in a vital pathway sequence, thus resulting in an inhibition of pea pollen growth.
Caffeine is a basic molecule, whose ability to accept protons are denoted by the lone pairs in the nitrogens' spa hybridized orbitals. Amazingly, only by one methyl group on a nitrogen does caffeine differ from theophylline; both drugs have similar pharmaceutical effects in the help of combating respiratory issues (Jellison et al., 1994). Also the most widely consumed drug in existence, it is estimated that 70 mg of caffeine is consumed per day by the equivalent of every single individual (including all children) in the world. In only 30 minutes after consumption, it is believed caffeine is completely absorbed by the human body. Therein, it goes to work having a great many effects -including the relaxation of smooth muscles (thus assisting in respiratory issues) and the stimulation of the nervous systems of humans and lab animals (Griffiths and Woodson, 1988).
Wishing to further these investigations, this study suggests that the introduction of 10 mL of caffeine solution over one week to the surrounding Miracle GrowÂ© soils of radish (Raphanus sativius) and wheatgrass (Tritican aestiuvum)-planted in flower-bed cells-will significantly inhibit each plant's growth and protein content: as will be determined on the seventh day of caffeine introduction by a Bradford Assay. To support this, 24 seedlings of 12-radish and 12-
wheatgrass were watered with 10 mL of distilled water every three days for a total of five waterings per plant, thus allowing the plants time to mature. (Herein "old" refers to six radish and six wheatgrass allowed to mature from week-1, and "new" refers to six radish and six wheatgrass allowed to mature from week-2) After this first two weeks, the remaining mature plants (12 - old wheatgrass (V), 12 - old radish (RÂ°), and 9 - new radish (Re)) were 10mL watered with a one of three caffeine-distilled water diluted solutions: three RÂ°, two Re, and three WÂ° were watered with a 0.25mM (0.01x dilution with pH of 5.97) solution; three RÂ°, two Re, and three WÂ° were watered with a 2.5mM (0.1x dilution with pH of 5.66) solution; three RÂ°, two Re, and three WÂ° were watered with a 25mM (lx dilution with pH of 5.98) solution. The remaining nine plants were watered with only pure distilled water to be used as a control group. After the watering period, each of the plants were harvested to have its protein content extracted, stem height measured, and fresh weight massed.
It was expected that those plants which received the highest caffeine addition would be the shortest plants with the least amount of protein content and smallest fresh weight as prior research would suggest, with the control subjects holding the greatest values for each variable. Statistical significance among the samples was collected via ANOVA tests that also allowed for the calculation of standard errors and means.
The findings of this study could prove vital to further investigation of caffeine's affects on growth and protein content within both animals' and plants' cells as the pathway in which the caffeine interferes can be more-so explicitly determined and further investigated. Due to caffeine's apparent similarity to theophylline, perhaps its use as a densely concentrated form could assist in the coping of patients with asthma or COPD, or, if the pathways are further investigated, perhaps the collaboration of caffeine with anti-bodies could prove vital in the
production of agriculture-stock that may waste valuable energy growing the unnecessary reproductive pollen tubes and thus would have less agricultural output.
Results and Conclusion (written by Sarahbeth Barlas)
After five weeks in, this study has continued to attempt at determining if the introduction of various diluted caffeine solutions (0.25mM, 2.5mM, or 25mM) will, in either radish or wheatgrass, inhibit stem growth and/or fresh weight and/or protein content as would be observed after harvest procedures on the seventh day of caffeine introduction. However, no purely visual differences could be seen between the control and treatment samples prior or at harvest time (Fig. 5).
In order to determine protein content significance, a Bradford Assay was constructed from Bovine Serum Albumin (BSA) with known concentrations of protein content. These protein contents' absorbances were taken by spectrometer for the construction of a Beer Lambert Law Standard Curve (Fig. 1 and Table 1 -experimental by David Maison, 2010). This plot resulted in a fairly strong R2 value of 0.9289. From this plot, each of the unknown protein contents of this study's samples were determined from the absorbances of homogenized 0.1g solutions (Table 2). The data analysis of ANOVA showed statistical significance only in the RÂ° samples with a p-value of 0.005 (Fig. 2). The lack of significance in Rn however, may imply that caffeine does not have an effect on radish growth until at least two weeks of treatment are conducted. To investigate further, it is suggested that a large sample size is enlisted (this study had only three plants for the radish groups and 2 for the old wheat grass). It is also recommended that more time is given for the drug to take full, statistically significant effect: perhaps more cellular or other internal development needs to mature before the drug shows any significance, and so time may be a valuable factor to consider. It is also interesting to consider that where the samples were taken from for homogenizing may have had an effect on significance: perhaps the caffeine only affects specific pathways in certain aspects of the plant
(e.g. the leaves verses the stem); wheatgrass-which did not show significance-is a much more leafy-plant than radishes, so it is hard to say whether any non-observed lack of significance is present due to lack of variables taken into consideration during this study.
At the time of harvest, plant stems' heights were measured by pulling up the plants by their roots and laying them along a ruler. Perhaps more accurate results could have been achieved for the wheatgrass that has a tendency to grow perfectly straight opposed to the radish's tendency to curl around itself, because this curling made it difficult to pull the stems taunt without breaking them. On the contrary, the measurements for the wheatgrass samples were taken from base of the roots to the base of top of the tallest leaf whereas radish measurements were taken from the base of the roots to up the base of the largest leaf (regardless of the largest leaf's size or length), so, again, if caffeine interferes with the pathways of proteins in only certain aspects of the plant, like the leaf for example, there may be more error in this data than is statistically relevant. In the end, statistical significance was only seen by an ANOVA test for WÂ°, which had a p-value of 0.036. However, it is suggested for further studies that plant leaf mass is taken into account.
Finally, the fresh weight was collected for each sample after recording stem height: the plants were each shaken and washed of excess soil and weighted on a balance. One concern herein was not being capable of removing all of the excess soils without loosing some root mass in the process or loosing root mass when removing the plants from the soil in the first place. Therefore, it is recommended for later experiments that such investigations are done without the inconvenience of soil particles that may unseeingly be left behind or the plausible loss of root mass in the soils upon harvest. In all, ANOVA tests showed significant difference for both the RÂ°'s and Re's fresh weights (with p-vales of 0.004 and 0.003 respectively). This is interesting
because the stem height WÂ° was shown to be affected by caffeine introduction (Fig. 4). Perhaps the leaves of the wheatgrass remained large (even though the stem height may have been inhibited by caffeine), and so V's fresh weight only appear to not be affected because it has very large leaves and very little stems naturally.
One major concern that was not taken into consideration prior to experimentation is that each of the flower-bed cells had little holes in the bottom of the cups. It is very likely that cross contamination between samples occurred due to leaking of solutions through these holes which could then be reabsorbed into the neighboring samples; for the reason, it is suggested that
samples be kept separate and fully contained from one another in future studies.
Contrary to the predictions, unusual results were seen: it was expected that caffeine's introduction would negatively affect the stem height, fresh weight, and protein content of each sample significantly over the control group, and why it did not is still unknown. It is thus recommended that further experimentation be conducted to determine which aspect of plant physiology is affected by the introduction of caffeine, and to continue investigations specifically within that subject area to compare between species with significantly different physiologies like those of the minimal-stemed wheatgrass and bulky-leaf radish.