Effects of Oil Contamination on Root Growth of Arabidopsis

1. Hypothesis Methods Introduction Discussion Results References Acknowledgements The Effects of Oil Contamination on Root Growth of Arabidopsis Matt Steelman, Josh Fester, Tinus Van Wyk, and Nicole SowersWofford College, South Carolina Some Arabidopsis ecotypes such as those from Ireland and Bulgaria grow on roadsides where petroleum runoff could affect their growth. In an experiment using phenanthrene as the pollutant, Arabidopsis plants were shown to be negatively affected 1. Our purpose is to investigate to what extent, if any, the root growth of Arabidopsis is affected by contamination with motor oil. In this experiment, we used ecotype Columbia because it is the most commonly used ecotype in laboratory studies. Also, we chose our concentrations of motor oil in agar with the desire to avoid killing the plant while providing adequate range that would produce an observable response. Germination Germination was unaffected by oil dissolved in the agar (p=0.667, χ2 with 6 degrees of freedom = 4.08, data not shown.) Firstly, germination was not affected by motor oil in agar, suggesting that environment is less important than conditions inside the seed for germination. Secondly, our results support the hypothesis that an increased concentration of motor oil in agar stunts root growth. Although there was no different in root growth from 0% oil to 0.01% oil, but at the higher concentrations (0.1% and 1%) root growth was significantly reduced compared to control. In general, root growth is negatively affected by increased concentrations of motor oil. Implications of this Experiment The results of this experiment show that while plants can tolerate low levels of motor oil pollution, increased concentrations are detrimental to root growth. It would be unwise to assume that all seedlings respond to oil pollution in the same way as Arabidopis thalania ecotype Columbia. It is likely that other ecotypes or other species of plants have greater or lesser sensitivity to oil in the soil. Future Experiments We recommend that future experiments find a more effective way to measure the root such as removing them from the agar. Also, the experimenter must make sure that light is evenly distributed to each agar plate. Another issue that should be addressed is clumping of motor oil in the agar which may allow roots to avoid the pollution by growing in the agar between the droplets of oil. Finally, effects of the motor oil on root growth in agar may be different than those in soil, so perhaps soil should be used in a follow-up experiment. Root Growth At the lowest concentration of oil (0.01%) root growth was not significantly different from the control group. However, root growth was decreased at the higher concentrations of oil in agar (p=0.001, Analysis of Variance FdF= 3, 70 = 6.054.) The addition of motor oil into the agar for Arabidopsis thaliana ecotype Columbia will significantly stunt the growth of the root. Root Growth and Oil Concentration Eight conventional Petri dishes containing 0.8% agar Castrol motor oil (5W30 ) Eighty Arabidopsis (ecotype- Columbia) seeds Image J Software Fluorescent grow lights Standard agar was mixed with motor oil to obtain 0%, 0.01%, 0.1%, and 1% dilutions. The agar containing motor oil required extensive mixing to disperse the oil before pouring on to slanted petri dishes. We then planted 10 seeds of Arabidopsis in each of eight Petri dishes; 2 dishes for each concentration . Seeds were placed at the upper edge of the agar on each of the plates. Agar was used rather than soil so that we could measure the roots without removing the plant from its substrate. The dishes were sealed and vertically placed beneath fluorescent grow lights at a distance of 33cm throughout the experiment. The two dishes with 0% motor oil in agar served as the control group, while the other six had the concentrations of the petroleum product. Each of the plates had equal photoperiods (16 hours of light and 8 hours of dark) 33 cm below a fluorescent light. Once a week for two weeks, we measured the length of the roots using Image J software (see picture below) from scans of each plate. We were able to accurately see and measure roots and shoots for individually identified seedlings. Only root measurement data are presented here.. 1) Lui, Hong. “An oxidative stress response to polycyclic aromatic hydrocarbon exposure is rapid and complex in Arabidopsis thaliana.” Plant Science: Vol. 176 Issue 3 March 2009 pg. 375-382. Special thanks to GR Davis and C. Abercrombie for their assistance with statistical analysis and answering of questions. Also, thanks to A. Steadman and T. Player for their comments and suggestions. Bars are 95% confidence intervals.

2. Summary & Status of Hypotheses Introduction Example of Petri Dishes Materials and Methods Hypothesis Germination Rate Discussion Acknowledgements Root Length of Plants under Fluorescent and LED lights Explanation of Graph Effects of LED and Fluorescent Lights on Root Growth of ArabidopsisKevin O’Quinn, Mesha Arant, Jordan Ball, and T.J. WhiteDepartment of Biology, Wofford College, Spartanburg, SC 29303 The germination rate, not shown, was not statistically different for seeds under fluorescent and LED light (Chi-square (dF=1)=0.137; p=.712). Different types of lights have been used to increase the growth of various plants. Our experiment tested whether LED or fluorescent lights produce plants with longer root length and higher germination rates. Fluorescent lights, which emit heat, consume more energy, and emit a more intense light are typically used in laboratories. LED lights, which consume 10% less energy than fluorescent lights, can last for many more years that fluorescent lights, but are more expensive. Are the LED lights really worth the cost? We performed an root growth experiment to find out. 1. Germination was not affected by light source. 2. Root growth was greater under fluorescent lights. Our results did not support our hypotheses. Our experiment showed that fluorescent lights produced plants with longer roots than the LED light panel. Of course, one of the drawbacks of fluorescent lights is that it requires more energy than LED lights to operate. This may be contribute to higher operational costs, but may have aided in the difference in root lengths between the fluorescent and LED lights. Because the fluorescent lights used more energy, perhaps that they emitted more energy. This extra energy could have given the plants grown under fluorescent lights the advantage. In comparison, because the LED lights used less energy, they emit less energy; this could have contributed to the shorter root length. Since the LED lights were less intense, we should have moved them closer to the plants in order to have the same intensity as the fluorescent lights. In the future, it would be best to figure out the distance from the lights to the plants in order to equalize the intensity being exposed to the plants. • Germination rates will be higher in plants grown under the LED lights. • Root length will be greater in plants grown under the LED lights. • Materials • 48 Arabidopsis seeds(Ecotype: Columbia) • Petri dishes with 0.8% Agar • Fluorescent and LED Growing Lights • Methods • 0.8% Agar was placed into 6 slanted Petri dishes. • Eight seeds were placed in each dish along the thin edge of the agar to reduce crowding. • Dishes were sealed and placed in a vertical position under the LED and Fluorescent lights at a distance of 34cm. On a photoperiod of 16 hours light and 8 hours dark. • Each seed was individually identified. • Germination rates and root length were measured weekly for two weeks. • Dishes were placed on a scanner and the images were analyzed with ImageJ software; root lengths were measured. The circles show the average root length and the error bars show 95% confidence intervals. Root growth from week 1 to week 2 was greater for plants grown under Fluorescent lights than for plants under LED lights (p=0.002, Chi Square Fdf=1,36=1.8. ) We did not graph the growth from Week 0 to Week 1 because during that interval growoth was likely produced by energy from the seed’s endosperm and not from the light. We thank G.R.. Davis for assistance with experimental design , Ab Abercrombie for help with statistical analysis, and A. Steadman and T. Player for helpful comments.

3. Methods Introduction Discussion References Shoot Emergence Results Acknowledgements Hypothesis Germination Results Does Seed Depth of Arabidopsis Affect Germination or the Ability to Breach an Agar Surface? Morgan Hiler, Carrie Martin, and Hannah LeirmoeWofford College, South Carolina These results support the original hypothesis that the seeds planted in both conditions would germinate; however, those seeds planted beneath the agar domes generally did not break the surface. Therefore, in the natural habitat of the Arabidopsis, if the seeds are planted rather than wind distributed the seeds may not be able to survive. This is because the seeds may not have enough stored energy to support the plant long enough for the shoot to break the surface. The seedlings that emerged from the dome’s surface had been inadvertently displaced, and therefore had a shorter distance to grow to breach the surface. The seeds that were directly beneath the center of the domes did not reach the surface. These results suggest that if the experiment had continued the plants may have eventually broken the agar surface, or died due to insufficient gas exchange. Our plants did not die in two weeks because unlike soil, agar is clear and the seeds may have been able to carry on photosynthesis whereas in soil they could not. The agar also allows for the shoots to respond to phototropism from the fluorescent lights rather than just geotropism, which is all the seeds could respond to if planted below the surface of soil. Of the 42 seeds planted beneath the domes of agar 7 failed to germinate, and of the 42 seeds planted on top of the agar 7 also failed to germinate. There was no significant difference in the germination rates in either treatment (P-value= 1, Fisher’s Exact Test). Arabidopsis seeds are wind distributed; they germinate and grow where they land in the surface of soil. In the laboratory to facilitate the measurement of shoot and root growth the seeds are placed on agar. As far as we know there have been no previous experimentation to determine if the seeds of Arabidopsis grow when planted beneath the surface of agar. Seed Placement Arabidopsis seeds will germinate as well under the agar as on top of the agar. The shoots of the Arabidopsis seeds planted beneath the agar will not break the surface. Week 2 Shoot Growth Of the forty-two seeds that were planted beneath the domes of agar four breached the surface. The other seeds grew sideways between the bottom of the agar dome and the top of the agar in the Petri dish. The shoots were beginning to grow leaves, but they were not able to grow straight up through the agar. Experiment Procedure: 1. Each of the 14 seeds, Ecotype Columbia Arabidopsis, spaced evenly across the surface of six Petri dishes filled with 25mL solidified 0.8% agar. 2. Make 42 domes, approximately ½ in in diameter, with 0.8% agar on a flat. 3. Allow domes a few minuets to solidify, then place them on top of seven seeds in each Petri dish. 4. Seal dishes and placed them under florescent lights for a 16 hrs light and 8 hrs dark cycle. 5. After a week check seeds with a dissecting microscope for germination. 6. After two weeks check seeds again with the dissecting microscope for germination, and if any shoots have breached the surface of the agar. Weigel, D. & J. Glazebrook. Arabidopsis: A Laboratory Manual. 2002. Cold Spring Laboratory Press. Thanks to Professors Davis and Abercrombie for their support, suggestions, and help with statistical analysis. Thanks also to T. Player and A. Steadman for their helpful comments.

4. Indroduction Materials and Methods Hypotheses Results: Effect of Agar Concentration on Germination Status of Hypotheses Discussion The Effects of Agar Concentration on Root Length in Arabidopsis thalianaMatthew Boggs, Aaron Seigler, and James SkinnerDepartment of Biology, Wofford College, Spartanburg, SC 29303 Results: Effect of Agar Concentration on Root Growth Agar, a gelatinous substance derived from red algae, is commonly used at a concentration of 0.8% to grow Arabidopsis plants in the laboratory. To determine whether this is the optimal agar concentration for root growth, we measured root growth in agar that was twice (1.6%) and half (0.4%) the standard concentration. TheArabidopsis thaliana ecotype Columbia was used for the experiment as it’s the most commonly used ecotype in the lab. • Agar composition did not significantly affect root growth in the Arabidopsis thaliana (Columbia). • The density of the agar solution did not affect germination rates. Agar concentration had no statistical effect on root growth at 2 weeks (p= 0.062, Repeated measures analysis of variance, DF=2, 29 F= 3.06) The 1.6% agar produced some of the longest roots, but statistical analysis showed no significant difference across the agar concentrations. The consistency of the 0.4% created an unstable substrate, such that measurements were difficult to obtain. While there was no significant differnce for any of the concentrations tested, given that the “p-value” approaced statistical significance, we feel that a repeat of the experiment would be appropriate with a larger number of seeds. Other possible experiments could include testing of even denser concentration to determine if density of the solution inhibits root growth. Nonetheless, we would recommend that the densities tested in future experiments should not be below 0.8% to ensure a stable substrate. 1. The greater the density of an agar solution that Arabidopsis thaliana (Columbia) seeds are cultivated in, the longer the roots. 2. The density of the agar solutions will have no effect on germination rate. Materials 6 round agar plates 48 Arabidopsis seeds ecotype (Columbia) 1 set of fluorescent grow lights 0.4% agar in two plates(Control Group) 0.8% agar in two plates 1.6% agar in two plates Methods Eight seeds were evenly spaced on upper edge of the agar in each plate. Dishes were sealed and marked. Dishes were placed vertically 33 cm beneath a lamp on a 16 hr light 8 hr dark cycle. Plates were scanned (below) and measurements of root length were made weekly for two weeks with Image J software. Note: bars are 95% confidence interval. Acknowledgements The authors are indebted to G. R. Davis for assistance with experimental design and Ab Abercrombie for assistance with statistical analysis. Agar concentration had no effect on germination rate (p=0.262, Pearson Chi Squared Test df2=5.259, data not shown.)