Introducing Excess Auxins to the Growth of Bean Seeds

Introduction: Perhaps one of the most common plants gardened today is the bean plant. Bean plants are dicotyledonous angiosperms, meaning they contain two cotyledons for the storage of starch. This starch is used by the plant during germination, before the growth of leaves can provide the plant with energy via photosynthesis. In order to successfully grow, bean plants require ideal conditions. In order to successfully germinate, a bean seed must have a sufficient amount of water and heat. Bean plants grow best in full sunlight, planted in soil that is loose and moist- with a pH of about 6.0. Like any other plant, a bean plant needs a variety of nutrients. The primary macronutrients that a plant needs are nitrogen, phosphorus and potassium. Nitrogen and phosphorus are components of a plants proteins, DNA and RNA; potassium is crucial in the process of up-taking water in a plants roots and also controls the operation of the stomata on the plants stem and leaves. The secondary macronutrients that a plant needs are magnesium, calcium and sulfur. Magnesium is a necessary component of chlorophyll, which a plant needs in order to photosynthesize; calcium strengthens the cell wall structure in a plants cells; sulfur is important in the production of protein and also promotes the activity and development of enzymes. The amounts of macronutrients that are present in a plant are crucial for the plants normal growth. Excess and deficiency of each nutrient will result in a variety of unhealthy effects that have several visual symptoms. Nitrogen deficiency in plants will result in a light green to yellow appearance of leaves (especially older leaves), stunted growth, and poor fruit development. Excess nitrogen in plants will result in dark green foliage which may be susceptible to lodging, drought, disease and insect invasion. The plants fruit and seed crops may fail to yield. Phosphorus deficiency in plants will result in possible purple coloration of the leaves, stunted plant growth and delays in development. Excess phosphorus in plants may cause deficiencies of micronutrients- especially zinc or iron. Potassium deficiency in plant will result in the older leaves turning yellow around the edges and dying, as well as irregular fruit development. Excess potassium in plants may cause a deficiency in the plants magnesium and calcium levels. With nutrients being regulated efficiently (no excess or deficiencies), the number of variables affecting the plants growth is fairly low. In this experiment, the nutrient levels were kept as neutrally equal as possible, eliminating the variable of nutrient levels. Two of the most important variables that affect the growth of a plant are external factors and hormonal levels. A plants growth is affected by a variety of external factors, the most important of which are light, water and temperature. In order to grow, a plant must obtain light to photosynthesize and make energy to sustain its growth. Water in plants is needed to fill cell vacuoles for strength; allow plant responses such as turgor pressure; as well as transport the necessary minerals the plant needs to survive. The temperature of a plants environment affects how well it metabolizes. If the temperature is warm enough, a

plant will show an increase in the effectiveness of photosynthesis- if the temperature is too cold, the effectiveness of photosynthesis will be reduced. In this experiment, the external factors above were controlled at a normal level: this eliminated main external factors as a variable in the bean plants growth. During a plants growth, a variety of hormones are involved. One main hormonethe hormone being used in this lab- is auxins. In regular concentrations, auxins promote cell elongation in the shoots and roots of the plant. Auxins are responsible for causing phototropism responses, stimulating the growth of tree buds and young shoots, and are responsible for the evolvement and maintenance of apical dominance. An excess concentration of auxins, however, causes the elongation of roots and shoots to be inhibited. Another important plant hormone is cytokinins. In regular concentrations, cytokinins stimulate cell plant growth via cell division. They reduce aging and stimulate the development of buds, as well as dark-germination of light-dependent seeds. At aboveaverage concentrations, cytokinins cause the growth of callus tissue; at below-average concentrations, cytokinins reduce root development. Gibberellins are the third main plant hormone. Gibberellins promote cell elongation as well as lateral growth in a plant. They stimulate the germination of pollen and the growth of pollen tubes, and are responsible for promoting growth in the embryo of a seed. Gibberellins induce the development of fruits to twice the normal size. In this investigation, excess auxins will be introduced to bean seeds during separate phases of their growth. Auxins in dissolved form and auxins in paste form will both be used, as well as a dissolved auxin powder/water mix.. By adding auxins to groups of bean seeds based on each of four pre-determined phases (germination, root growth, shoot growth, all) and comparing the qualitative and quantitative results to those of the control group, a higher understanding of how drastically auxins affect plant growth is to be obtained. It is predicted that excess auxins will affect the growth of bean seeds by: slowing down the germination process; hindering the growth of roots; and inhibiting the height of the shoot. This is predicted to occur because excess auxins are known to inhibit cell elongation in the roots and shoots of plants. By inhibiting the elongation of these major cells, auxins will ultimately cause a lack of growth in the bean plant.

Materials: Glass beaker containing dissolved auxin/alcohol solution* Glass beaker containing auxin paste solution* Glass beaker containing dissolved auxin/water solution Volumetric flask 25 plastic planters 25 bean seeds Regular soil Ruler Popsicle stick 5 Petri dishes

Paper towels Auxin powder Camera device

* It is important to note that the auxin solutions were obtained from the teacher, who prepared them using the following instructions: 1. Dissolve level tsp. of IBA in tsp. of grain alcohol 2. Stir mix into 5qt of water to make a solution with a concentration of 0.01 3. Mix 1 tsp. of solution in 1 tsp. of melted lanolin 4. Mix paste thoroughly Procedure: To begin the investigation, twenty-five bean seeds and five Petri dishes were obtained. The Petri dishes were then labeled, Germination, Roots, Shoots, All, and Control before they were all- except for the All and Germination- filled with tap water. In the All and Germination Petri dishes, a modest amount of distilled auxin/alcohol solution was added. Five of the twenty-five bean seeds were then placed in each dish. After a sufficient amount of time had passed, twenty-five plastic planters were readied by labeling them, Germination, Roots, Shoots, All, and Control, in groups of five. The five bean seeds from each Petri dish were then removed from their Petri dish and placed in paper towels, specified by their labeled grouping, moistened with water. This was to allow each bean seed to have a larger supply of oxygen than semi-submergence in Petri dishes would allow, while still absorbing water for the germination process. When enough time had passed, each bean seed was removed from the paper towels and plantedat the depth of one fingertip- into their own planter, with their respective labels. A volumetric flask was filled with water and an equal amount of water was poured into each planter. The watering of each plant continued each day until the first soil-penetrating shoot became visible on the seventh day. At this point, it was determined that germination had occurred and the next steps could be completed. A solution of dissolved auxin powder and water was then poured into each Roots planter. The dissolved auxin powder and water solution was used due to suspicion of the alcohol (in the dissolved alcohol/auxin solution) causing possible death of the ten seeds that were not soaked in water. Each plant was then watered with an equal amount of water from the volumetric flask. Again, the watering of each plant continued each day until the thirteenth day, when the Shoots planters contained plants with shoots growing. Using a popsicle stick, a moderate amount of auxin paste solution was then applied to each of the two growing Shoots plants. Each plant was again watered with an equal amount of water and the growth of each plant was recorded by taking photographs from a cellular phone. Each plant was watered each day until the end of the investigation. Each day, the plants with shoots present were rotated to reduce the effect of phototropism. Measurements

and photographs of each plant with shoots present were obtained on the sixteenth day of the investigation, and then again on the twentieth day and the twenty-fifth day.

Observations: Upon returning to the beans soaking in the Petri dishes to begin germination, it was evident that the beans soaking in water had absorbed a substantial amount of water, due to the amount of enlargement that had occurred. The beans soaking in distilled auxin/alcohol solution had not shown any change in size. The colour of the beans in the auxin solution was a slight orange tint, whilst the beans soaking in just water remained the same white colour. When each bean seed was allowed to sit in the moist paper towels, every seed increased in size- showing that water absorption was still occurring. On the fifth day of the experiment, one Control plant had begun to grow out of the soil. The hypocotyl had penetrated the soil enough to allow the plants cotyledon to show. One Roots plant had also begun to grow out of the soil. Though the hypocotyl had penetrated the top of the soil, the cotyledon was not yet visible. On the seventh day of the experiment, two of the Shoots plants had showed their hypocotyls penetrating the soil. One of these two plants cotyledons (that of Shoots plant 1) had become visible while Shoots plant 2s cotyledon was not yet visible. The cotyledon of the one growing Roots plant had become visible on a short shoot. No growth from any of the Germination or All beans was present. On day eight, a similar height between the one growing Roots plant and the Control plant was present. There was no growth present in any of the planters containing seeds that were initially soaked in the dissolved auxin/alcohol solution. On the twelfth day of the experiment, each of the growing plants (one Control, one Roots, two Shoots) had cotyledons above the ground. The Control plants first true leaves were nearly completely developed and the Shoots plant 1s first true leaves were beginning to grow away from the cotyledon. No other plants showed any signs of first true leaf growth occurring.

Table 1.0 Height of Bean Plants (Day 16)

Height of Bean Plants (Day 16)

Ground to Cotyledon (cm) 2.5 4.5 1 6.5 Total Height (cm) 2.5 11 3.5 "Roots" 19

20 Plant Roots Shoots plant 1 15 Shoots plant 2 Height 10 Control (cm) 5

Table 1.1

0 Ground to Cotyledon

Total Height

"Shoots" 1 "Shoots" 2 "Control"

Measured Range

The sixteenth day of the experiment showed many results. Firstly, (as seen in Table 1.0 and Table 1.1) the height of the Control plant- which was the same age as every other plant- was much more than that of any other plant. The total height of the Roots plant was 2.5cm, which was 16.5cm less than that of the Control.

Figure 1.0 Shoots Plant 1: First True Leaves

Figure 1.1 Shoots Plant 2: First True Leaves

Figure 1.2

Control : First True Leaves

Shoots plant 2, which had been covered with the auxin paste solution just three days previously, had begun to show its first true leaves. Unlike the true leaves present on the Control plant (refer to Figure 1.2), these first true leaves were starting to grow almost instantly out of the cotyledon, without any shoot growth occurring between the cotyledon and the first true leaves (refer to figure 1.1). It became evident that the first true leaves of Shoots plant 1 were also growing very close to the cotyledon (refer to Figure 1.0), as opposed to those of the Control plant.

Table 1.2 Height of Bean Plants (Day 20) Plant Roots Shoots plant 1 Shoots plant 2 Control Ground to Cotyledon (cm) 2.5 5 1 6.5 Total Height (cm) 2.5 11.5 4 37

Table 1.3

Height of Bean Plants (Day 20)

40 35 30 Height 25 20 (cm) 15 10 5 0 Ground to Cotyledon

Total Height

"Roots" "Shoots" 1 "Shoots" 2 "Control"

Measured Range

On the twentieth day of the experiment, the measurements of each plant once again revealed interesting observations. The Roots plant did not change in height at all during the four days. The growth that occurred in both Shoots plants was extremely small, producing a height change of only 0.5cm in each of the two plants. The Control plant however, did continue to grow. In the four days that passed since the last measurements, the Control plant grew from 19cm to 37cm, for a growth of 18cm- nearly double its previous size (refer to Table 1.2 and Table 1.3).

Figure 1.3 Control: First Leaf Texture & Shape

Figure 1.4 Shoots Plant 2: First Leaf Texture & Shape

Upon examining the shape and texture of the first true leaves of the plants, it was evident that the leaves of the Control plant were very different from those of either Shoots plant. The leaves of the Shoots plant 2 are a much darker green, in some places, than those of the Control. The first true leaves of Shoots plant 2 (refer to Figure 1.4) are smaller and more brittle than those of the Control, which were large and smooth (refer to Figure 1.3). There were also several tiny bumps present on the surface of the leaves in Shoots plant 2, which were not present in the leaves of the Control. Figure 1.5 Roots Plant Death

On the twenty-third day of the experiment, the Roots plant was found dead and rotting. Upon inspecting the shoot, which had not shown growth for over seven days, the entire plant was found to be very dry. The shoot of the plant was very thin and string-like; the cotyledons were split open and browning, and were empty. Table 1.4 Plant Roots Shoots plant 1 Shoots plant 2 Control Table 1.5 Height of Bean Plants (Day 25) Total Height (cm) 0 12.5 5 53

First True Leaves to Second True Leaves (cm) 0 4 1 11

Height of Bean Plants (Day 25)

60 50 40 Height (cm) 30 20 10 0

First T.L to Second

Total Height

"Roots" "Shoots" 1 "Shoots" 2 "Control"

Measured Range

On the twenty-fifth day of the experiment, the final measurements showed interesting data. The Roots plant had died and so could not be measured for growth ( refer to Figure 1.5). The Control plant continued to grow at a steady rate, growing from 37cm tall on the twentieth day to 53cm tall on day twenty-five (an increase in 16cm). (Refer to Tables 1.4 and 1.5) These measurements were compared to the height change in Shoots plant 1 from day twenty to day twenty-five (an increase in only 1cm) and that of Shoots plant 2 (an increase in only 2cm) and it was found that the bean plant grew at least eight times higher than any plant with excess auxins added to the shoot. Figure 1.6 Control: Distance Between First True Leaves and Second

Figure 1.7 Shoots Plant 2: Distance Between First True Leaves and Second

It was interesting to see that the second set of leaves that occurred after the first true leaves did not always spread out correctly. In the Control plant, a distance of 11cm (refer

to Table 1.4 and Figure 1.6) separated the first true leaves from the second set of leaves. In the Shoots plant 2, a distance of only 1cm (refer to Table 1.4 and Figure 1.7) separated the first true leaves from the second set of leaves.

Discussion: The germination process that occurred in this experiment yielded only four growing seeds- out of the possible twenty-five seeds that were started with. The fact that only two Shoots seeds successfully germinated, only one Control seed successfully germinated and only one Roots seed successfully germinated was not largely human error. Even if every situation were ideal for a seed to germinate and successfully become a living plant, instances occur when the seed fails to do so- its nature. However, in this experiment, many of the failed seed germinations could be explained. Firstly, although unknown at first, alcohol tends to be a growth retardant in plants. The fact that alcohol was one of the ingredients used to make the dissolved auxin/alcohol solution that ten seeds (five Germination and five All) were initially soaked in may be the reason none of theses seeds successfully germinated. One reason why any of the seeds that were initially soaked in water did not germinate may be that the amount of water they were soaking in was too much- which caused too much water absorption or a lack of oxygen. The Roots plant, which happened to be one of the first plants to have its cotyledon visible above the soil, was alive and well until the dissolved auxin powder/ water solution was added. Once the solution had been added, the plant showed no growth and eventually died. This may be because the plant did not have its first true leaves growing when the auxin solution was added. When the solution was added, it may have caused the shoot and epicotyl cells to stop elongating and growing, thus stopping the first true leaves from ever appearing. Since no true leaves were present, the plant could not obtain energy via photosynthesis and had to use the energy left in the cotyledon. Once the starch in the cotyledon had been used up, the Roots plant would have no other way to obtain energy and would therefore eventually die. The effects of the auxin paste solution on the shoot of the Shoots plant were drastic. Not only did the solution cause reduced growth- as expected- but it also caused darker leaf colour in spots; smaller, more brittle leaves; bumpy spots similar to callus tissue on the leaves; and shorter distances between the first true leaves and the second set of leaves. The reduced growth caused by the auxin paste solution almost certainly occurred due to the cell-elongation-inducing effects of excess auxins. Perhaps the darker leaves were a result of excess nitrogen, somehow associated with the excess auxins. For example, excess auxins induce the elongation of cells: this could somehow cause a mutation or deletion of DNA in the cells that are not growing properly- which could cause excess nitrogen because it is no longer needed in the DNA that is unused. The short distances, between the first true leaves and the second set of leaves, that occurred in the Shoots plants is very interesting. Somehow, the growth of the shoot after the first true leaves was hindered- due to induced cell elongation- while the growth of the second set of leaves, though also hindered, was able to occur. The second set of leaves

produced on each Shoots plant was much smaller than that of the Control plant, thus cell elongation must have been induced during the production of these leaves. If this experiment were to be performed again, it would be interesting to see how the same exact procedure could be incorporated into the use of cytokinins- which cause growth by cell division (the opposite of auxins). Conclusion: This experiment investigated the effects of excess auxins on the growth of bean seeds and plants. This was done by adding excess auxins to each main phase of a plants life: auxins were added to the germination process; auxins were added to the growth of roots; auxins were added to the growth of shoots; and auxins were added to each of the phases. These results were then compared to those of a control plant- which had the normal amount of auxins- in order to determine what the effects of auxins are on a bean plant. The results of the experiment showed that excess auxins in the germination phase (as well as excess auxins in all phases continuously) caused the inability for a seed to germinate. Excess auxins in root growth may cause premature death- especially if no true leaves were present before the auxin solution was added to the roots. Excess auxins in shoot growth caused a variety of instances: reduced plant height; darker leaf colour in spots; smaller, more brittle leaves; bumpy spots similar to callus tissue on the leaves; and shorter distances between the first true leaves and the second set of leaves. During the experiment, several possible sources of error may have occurred. Primarily, errors could have occurred in the germination of the seeds. In the seeds that were to be given excess auxins, the alcohol in the auxin solution used could have caused each of the seeds to die. To reduce this risk, an auxin solution using only auxin powder and water should be used. If the auxin powder and water solution had been used for the germination of the seeds with excess auxins, instead of alcohol, some of the seeds may have survived and germinated- which would have given extra data as to how the effects of excess auxins in germination affect the rest of the plants growth. During the germination of the seeds in just water, the amount of water each seed was in may have been too much. This may have caused too much water to be absorbed, or not enough oxygen for the seeds- resulting in their death. If this experiment were to be performed again, each of the regular germinating seeds should be germinated in moist paper towels only, to avoid the effects of too much water. Had the seeds only germinated in moist paper towels during this experiment, many more may have survived- which would have provided the experiment with more subjects- allowing averages of height to be obtained instead of single readings, resulting in increased accuracy To further this investigation, the effects of excess auxins could be taken to the sexual reproduction level. It would be interesting to see how the effects of excess auxins will affect the normal reproduction factors of a bean plant. For instance, would the hindrance of height in the plants- caused by the excess auxins- or excess auxins in general, result in delayed reproduction times in bean plants. To do this, the Control bean plant would need to be mixed with another healthy control bean plant so the conditions of the bean plants sexual reproduction are known. Then, the plants affected by excess auxins

could be mixed with a healthy control bean plant to ensure proper pollination, and the results of each test could be extensively compared.

References

Fertilizers and Plants. (n.d.). Retrieved from University of Arizona website: http://ag.arizona.edu/pubs/garden/botany/environmental.htm Iannotti, M. (2011). Green Beans - How to Grow Bean Plants in Your Garden. Retrieved from About.com website: http://gardening.about.com/od/vegetables/p/GreenBeans.htm Lang, S. S. (2006, March/April 31). Why tipsy flowers dont tip over: Booze stunts stem and leaves, but doesnt affect blossoms, study finds. Retrieved from Chronicle Online website: http://www.news.cornell.edu/stories/March06/drunk.flowers.ssl.html Plant Hormones. (1999). Retrieved from Oregon State University website: http://extension.oregonstate.edu/botany/hormones.htm Plant Nutrients. (2001). Retrieved from North Carolina Department of Agriculture website: http://www.ncagr.gov/cyber/kidsworld/plant/nutrient.htm Seed Germination. (n.d.). Retrieved from University of Waterloo website: http://retirees.uwaterloo.ca/jerry/orchids/nutri/html