Research Proposal
Sammie Cline, Alyssa McMahon, Zach Schmidt, Madi Traverse
Specific Aims
Our aim is to test the effect of increasing the salinity of a water supply on the health and boltheight of Arabidopsis thaliana . A secondary goal is to determine whether RNA interference continues to function in plants with various genes important to interference inactivated.
Background
Why Arabidopsis?
Arabidopsis thaliana provides many advantages to plant researchers, including a small, sequenced genome, ease of genetic manipulation, and a short life cycle. Because of its usefulness and its close similarity to many other plants, A. thaliana is considered a model organism in biological research. The Arabidopsis genome was the first plant genome to be sequenced; since
2000, scientists have had a record of about 115 million base pairs (Mbp) out of the 125 Mbp that comprise A. thaliana
’s genome. Knowledge of this genome allows researchers to determine where the plant’s 26,000 genes are located, and how their expression is related.
Genetic manipulation is relatively easy in A. thaliana ; in order to induce transformation in the plant, biologists often just dip it in a 5%-sucrose solution containing a small amount of detergent and the bacterium Agrobacterium tumefaciens . This bacterium injects T-DNA (transfer
DNA) into plant cells using plasmids, so transformation of Arabidopsis cells is a matter of inserting the desired DNA sequence into A. tumefaciens ’s Ti plasmid, which contains its T-DNA.
The bacterium then injects the plasmid, which in turn inserts the T-DNA into the plant cell’s genome by recombination. Using vectors like the Ti plasmid, scientists can also experiment with
“knocking out,” or inactivating, specific genes. Disrupting a gene with T-DNA typically causes that gene to code for a non-functional protein, effectively nullifying it. Other methods allow target genes to be completely excised from the genome. By comparing knockout specimens of
Arabidopsis to control specimens, scientists can determine the functions of the inactivated genes.

Many knockout varieties of Arabidopsis exist; about three-quarters of the plant’s genes have been inactivated across all of its knockout-types, and a knockout organism may have more than one gene knocked out. The DCL 234 and RDR 256 types used in class, for instance, have both had three of their genes inactivated.
Also useful is A. thaliana ’s six-week life cycle. Because an entire experiment with the plant can be performed in just 1.5 months, scientists can gather data quickly and move on to offspring generations from the initial experiment when studying cross-generational variables like epigenetic factors. The speed with which Arabidopsis plants mature and reproduce also allows researchers to rapidly design new experiments to explore observations made in the initial round; they do not need to collect data for years to be sure of their results. A. thaliana is small, making it useful for experiments in which space is limited and reducing the expense of plant maintenance. It also typically self-pollinates, which can help to ensure that a line is true-breeding for particular traits. A large number of filial-generation plants can come from a single individual; plants usually produce thousands of seeds each.
RNAi
RNA interference (RNAi) is the process by which particular mRNA transcripts are destroyed before being translated into proteins, effectively “turning off” the gene from which those mRNA sequences were transcribed.
Experimental Methods & Analysis
Rationale for Variable
We chose salinity as our variable because ecosystems across the world fall victim to disasters that alter the salt content of their water and soil. Tsunamis, for instance, carry salt water miles inland. Besides disasters, rising sea levels can also lead to an increase in salt content not only in surface water, but in groundwater as well. The plant populations of affected areas often include crops, so the economic impact of a salinity change could be disastrous depending on how much

salinity those crops can tolerate. As a result, determining the influence of salinity on a model plant like Arabidopsis is important.
Protocol
Planting: To plant the Arabidopsis we filled each of the pots up to the lip with moist soil and flattened each, making sure it was not to compact. Then we added a couple of seeds with a dropper to each corner of each pot. There are twelve pots, and for each of the three types of
Arabidopsis there are four pots.
Applying a Variable: Our variable is salt concentration in the soil. First we measure out one liter of water (with a beaker), then put the water into a clean milk carton. In the milk carton we add ten milliliters of salt (with beaker) and one scoop of fertilizer. We mix by shaking the carton.
The new water is applied to the experimental plants in a separate tub while we use normal watering for the control plants.
Collection of Data: To collect data we are looking at color changes with a disposable camera, making sure to have the same lighting with each (throughout experiments). This is a qualitative form of data just to compare the controls to the experimental plants. We are also measuring the height of the bolt from the rosette by carefully propping it up and measuring in millimeters. This will allow us to see if our variable is affecting the reproductive abilities of the plants.
Rationale for Data Collection
Color change, our qualitative measurement, could be a sign that the plant is under stress, thus confirming that the salinity difference between the control and experimental plants is a stimulus to which the plant is responding. Our quantitative measurement, bolt height, will be the true indicator of the increased salt content’s effect on the plants; if the bolts of the experimental plants do not grow as tall on average, the salinity has negatively impacted them, whereas if they grow taller it has beneficially impacted them. As it is likely to negatively impact them, the real question is how much of an effect it actually has.

Significance of Proposal
For our research project, our group will be focusing on the effects of salinity on
Arabidopsis thaliana columbia. Although salt (NaCl) is present normally in soils, adverse amounts of salt can result from many different natural events, affecting overall plant growth. Soil salinity is often changed by two major natural events: drought and tsunamis. Although these two occurrences have immediate effects on plants such as lack or excess water as well as physical strain and changes in grazing by animals, our group will be focusing on the latter effects caused by increased salt content, even after water levels have returned to normal.
Of course drought and tsunamis typically affect very different regions of the United
States and thus affecting different varieties of plants. While drought is typically centralized along the plains region, tsunamis and hurricanes have a greater stance in coastal areas, mainly the lower portions of the country. Although they occur in different places though, they produce much of the same lasting effects on the salinity of the soil that experience them. Through our experiments we will calculate the effects of salinity on Arabidopsis by taking different plant measurements, both qualitative and quantitative, as well as observing the differences presented in our two categories of mutants to see if either mutation promotes or decreases salinity tolerance in the plants. The relevance of these experiments can be seen in agricultural crop operations that are often effected by drought or surges of sea water and may provide the first steps in controlling crop losses due to these disastrous events.