Greenhouse Experiments

Results of Corn and Rice Control Growing — iGEM 2014

The purpose of a control group of corn and rice plants was to characterize the growth and chlorophyll parameters with respect to iron availability in the soil. We accomplished this by applying varying amounts of iron chelate—a synthetic iron supplement commonly applied to soils— in order to see how different levels of iron uptake affect plant growth. The ultimate goal is to compare the control data to the experimental data involving the application of our modified soil bacteria Bacillus subtilis.

Our first experiment consisted of 102 total corn and rice plants grown in the greenhouse for 4 weeks in order understand the growing process, and to collect some preliminary control data to compare to the round of plants treated with our Bacillus subtilis bacteria. We collected data for height, chlorophyll content, and final biomass. In measuring height, we measured from the base of the stalk nearest the soil to the top of the first leaf. The chlorophyll content was quantified with an Opti-Sciences chlorophyll meter, which measures chlorophyll content without damaging or removing any leaf tissue. We used corn leaf number 5 (see diagram), and measured the tissue one-third down from the tip of the leaf, as well as one-third up from the base of the leaf. At the end of the 4-week growing period, we removed all the plants at the base of the stalk and measured the final biomass.

The set-up of the experiment consisted of 17 sets of plants, each set with a different concentration of applied iron chelate, and three replicates for each set. An important aspect of this project was controlling plant nutrients in order to see the effect on the nutrient of interest. We used a standard nutrient solution, Hoagland’s solution, which contains all essential plant nutrients and is applied via watering. A standard peat moss soil medium was used because it is essentially neutral and is an easier medium in which to grow corn and rice than a sand or pure hydroponic system.

The iron application levels were derived from the Sprint 330 iron chelate manufacturer-recommended dose based and our biweekly application of nutrient solution. The 100% iron control plants received the manufacturer-recommended application, and the rest of the applications were percentages of that amount. These ranged from 0%-150% of the recommended dosage, which was broken down into 10% increments. Of the 17 sets, two were controls. The first control was watered only with nutrient solution and no iron application. The second control was watered only with deionized water, no Hoagland’s nutrient solution or iron chelate.

After planting the corn and rice seeds, over 90% of the corn seeds germinated within a week of planting. The rice plants took longer to germinate and less than half of them had germinated by the end of the four weeks. Based on visual observations, many of the corn plants looked very similar in terms of height and color. The phenotypic gradient we were attempting to create was not as drastic as we had anticipated. For example, the corn plants treated with 10% iron chelate solution versus those treated with 120% iron chelate solution did not look as drastically different as expected. However, with closer examination, differences could be seen between the varying iron levels in each set of plants. One major sign of iron deficiency in plants is chlorosis, which is the yellowing of leaves caused by the inability to produce chlorophyll. In corn, chlorosis manifests itself visually through yellow striping on the leaves.

At the end of the 4-week growing period, the results were both surprising and expected. There was an obvious difference in physical appearance of corn plants supplemented with 150% iron chelate solution compared to those with no iron treatment—the final average chlorophyll readings were 12.35 and 8.77 respectively (Figure 1). However, the difference was harder to tell between groups that were relatively close together, so the iron applications did not manifest themselves in an observable manner except in the case of the extremes. Additionally, the plants with low iron applications appeared to be healthy, with the exception of slight iron chlorosis, regardless of their lack of iron. This may be due to the fact that the soil medium had a slight base level of iron that the plants were able to use, but consumed quickly over the course of the 4 weeks. If time permitted, it would be expected that if the corn plants without an iron application were allowed to grow for 8 weeks, the chlorosis would be much more pronounced because the plant would have completely depleted the initial iron content that the soil medium contained. Contrastingly, the plants treated with 150% iron chelate solution appeared to have stunted growth, and their height measurements were significantly less than the corn plants grown with a lower iron application (Figure 2 and 3). The P-value of the height difference between most and least iron added was 0.011742, which is a significant difference. This was a good illustration of the adverse effects of excess iron in the soil.

Ultimately, the trends were as expected, except for a few outliers (see graphs below). We used this data in order to decide which iron concentrations to incorporate into the next round of planting—0%, 50%, 100%, and 150%. This will allow for range of iron efficiencies to compare to the plants treated with bacteria. This iron uptake data provided a base of what to expect when growing corn and rice with our modified bacteria. Because the bacteria are using the same iron-fixing mechanism that the plants themselves use, the bacteria will act as a natural iron supplement, utilizing all necessary iron in the soil. An example of the desired result would be a plant treated with 100% recommended iron application, which represents the ideal amount of iron added to the plant system, having the same health and growth pattern as a plant treated with our modified strain of Bacillus subtilis. We now know what to expect from our plants in the greenhouse and are able to compare these results to plants treated with bacteria to determine the effectiveness of our iron-uptake construct.

Figure 1 

Figure 2

Figure 3