Gibberellic acid belongs to one of the five most common categories of plant hormones: gibberellins. Gibberellins are known to stimulate plant growth and development, and gibberellic acid is the most common form of gibberellins. Chemically, gibberellic acid is a tetracyclic, diterpenoid compound, meaning that one molecule of GA3has four rings of atoms, twenty carbon atoms, and four methyl groups (Gupta & Chakrabarty, 2013). Gibberellic acid has long been used in agriculture due to its properties as a plant growth regulator. Gibberellic acid is known to induce seed germination, promote shoot growth and internode elongation, determine the sex expression of a plant, and it is involved in promoting the flowering of plants (Gupta & Chakrabarty, 2013). Gibberellic acid can be applied to plants in a variety of ways, from spraying an aqueous form onto the plant, to growing plants in a media containing the hormone, to dipping the plants into a gibberellic acid paste. In a 1956 study, researchers examined the effects of gibberellic acid on forty-nine different plant species. They used a variety of different techniques, such as spraying the plants with gibberellic acid, soaking the plants in gibberellic acid, and dipping the plants in gibberellic acid to expose the plants to the hormone. While the researchers established that plant responses varied greatly from species to species, it was clear that gibberellic acid promoted stem elongation. The researchers also found that the stem growth was the greatest in those plants that had just begun elongation (Marth, Audia, & Mitchell, 1956). The researchers did not test the differences between their application methods of gibberellic acid, however, and that extension will be investigated in this study. An additional study, conducted in 1967, looked at the effects of gibberellic acid on Solanum tuberosum growth and tumor formation. The plants that were treated with gibberellic acid grew taller and had a larger dry weight than the controls did. An unexpected change that the researchers noticed was that the plants treated with gibberellic acid became quite pale in color. The gibberellic acid treatment also led to an increased rate of cell division. Upon further inspection, researchers noted that the internodes of gibberellic acid treated plants had greater numbers of cells than in the internodes of the control plants. In addition, they observed that the gibberellic acid treatment resulted in a delayed development and smaller size of the tubers, but larger stolon size (Lovell & Booth, 1967). As a potential extension of this study, it would be interesting to test the effects of gibberellic acid on another species: Arabidopsis thaliana. Arabidopsis thaliana is a commonly used model organism in biology. It does not require a large amount of space to grow, as many plants can fit within one small petri dish. In addition, thaliana has a short germination period of only a few days, and a six-week life cycle, making it easy to study development. Lastly, there are many mutant strains of A. thaliana available for purchase and study (NIH, n.d.). One of these mutants, a plant with a mutation in the PKL 1-1 gene (commonly referred to as PICKLE) will be investigated in this study. This mutant is phenotypically similar to gibberellic acid-deficient plants, with its short stature and petioles. PKL1-1 is thought to code for a CHD3 protein that is a chromatin-remodeling factor. CDH3 is involved in repressing embryonic transcription and therefore decreasing plant growth. In a study conducted by the creators of the mutant, it was determined that gibberellic acid application can slightly reduce the effects of the mutation. Although the plants that researchers treated with gibberellic acid did not return to the same size as the wild-type plants, their size did slightly increase (Henderson et al., 2004). In this study, different methods of application of gibberellic acid will hopefully increase this growth even more. In this work, A. thaliana will be used to investigate the effects of varying methods of gibberellic acid exposure on the primary growth and development of both wild-type and PICKLE mutants. Three different concentrations of gibberellic acid will be applied to the wild- type plants either through spraying or by growing the plants in a gibberellin infused media. Whichever method of gibberellic acid exposure is the most effective in the wild-type plants will then be applied to the PICKLE mutants to attempt to reverse the effects of the gene mutation. The shoot and root lengths of the plants will be analyzed. My research question is: “Which method of gibberellic acid exposure, an aqueous spray or media infusion, is more effective in increasing the root and shoot growth of Arabidopsis thaliana, and at which concentration? Will applying gibberellic acid to PKL1-1 mutant plants reverse the effects of the mutation?” As for my hypothesis, I predict that the plants sprayed with the gibberellic acid will have more growth in the shoots than the plants grown in the gibberellin infused media, because when autoclaving the media components, I believe that the gibberellic acid may decrease in its efficacy. As for the different concentrations, I predict that the high concentration of gibberellic acid will have the most positive effect on the shoot growth of the plants. For the second part of the experiment, I predict that gibberellic acid exposure will increase the shoot growth of the mutants. However, I predict that gibberellic acid will have little effect on the root growth of the plants in all trials due to the fact that the hormone is traditionally used to impact stem growth. Materials and Methods:The experimentation was divided into two major parts: determining the method of gibberellic acid exposure that has the most positive effect on plant growth, and then attempting to reverse the PKL1-1 gene mutation using the most effective method from part 1. For part 1, several bottles of MS agar were prepared to serve as a growth medium for the Arabidopsis thaliana. In order to do so, 0.4 grams of MS salts, 1 gram of sucrose, and 0.8 grams of plant agar were measured using an electronic balance and poured into a media bottle. 100 mL of distilled water was also added to the bottle. The bottle was placed on a magnetic stir plate for two minutes and was then autoclaved. This process was repeated 5 times for a total of 5 bottles of normal MS agar. Additional agar infused with gibberellic acid was also prepared. The same procedure was used as above, but in one bottle, representing the low concentration, 385 µLof gibberellic acid stock solution was added (to create agar with a concentration of 50 mg/L). In the bottle representing the medium concentration, 770 µLof gibberellic acid stock solution was added (to create agar with a concentration of 100 mg/L). In the third bottle representing the high concentration, 1540 µLof gibberellic acid stock solution was added (to create agar with a concentration of 200 mg/L). A p1000 micropipette precise to multiples of 5 microliters was used to measure the gibberellic acid. 20 petri dishes of normal agar were poured, with 5 extras. 5 of these plates represented the negative control, 5 represented the plates that would be sprayed with the low concentration of gibberellic acid solution, 5 represented the plates that would be sprayed with the medium concentration of gibberellic acid solution, and 5 represented the plates that would be sprayed with the high concentration of gibberellic acid solution. In addition, 5 plates of each of the three concentrations of gibberellic acid infused agar were poured. The plates were inverted and stored in the refrigerator until use. The wild-type Arabidopsis thaliana seeds were placed in a microtube and bleached with 500 µLof 30% bleach solution. After a 3-minute soak, the seeds were rinsed with sterile water, stored in sterile water, and refrigerated until use. The seeds and prepared petri dishes were then taken into the tissue culture laboratory, and sterile technique was used to plant five seeds on each of the plates, as shown in Figure 1.  The plates were then placed under the plant lights. After three days, the plates designated for gibberellic acid solution sprays were brought into the tissue culture lab. 10 µLof the corresponding gibberellic acid solution (either 50 mg/L, 100 mg/L, or 200 mg/L) was pipetted directly onto each plant using a micropipette precise to the nearest microliter, to mimic the action of a spray. All of the plates were then parafilmed and placed under the plant lights. The following day, the plates were placed vertically in Tupperware containers divided into sections with lab tape underneath the plant lights. This allowed for greater shoot growth, because if the plates were kept horizontally, the shoots would grow up and hit the lid of the petri dishes. Beginning one week after planting, the root length and shoot length of each plant was measured over the course of four days using a metric ruler, precise to the nearest millimeter. The ruler was held against the transparent petri dish to measure both the roots and shoots. Color was used as a means of differentiating the roots from the shoots; the roots were a translucent white color and the shoots were green. Statistical analysis was conducted to determine the most effective gibberellic acid treatment-whether by introduction directly into the growth media, or by a gibberellic acid soak post-germination, and at which concentration. Part 2 of the experiment was entirely dependent on the results from part 1. In order to have the greatest likelihood of the mutants growing larger due to the gibberellic acid, only the treatment that had the greatest effect on the wild-type plants was administered to the mutants. The results are analyzed in further in the results and discussion sections, but for the purposes of this procedure, low and medium concentrations of gibberellic acid infused agar resulted in the most significant increase in plant growth. Thus, for part 2 of the experiment, 2 bottles of normal agar, 1 bottle of low concentration gibberellic acid infused agar, and 1 concentration of medium concentration gibberellic acid infused agar were prepared and poured into 20 petri dishes, as designated on Figure 2. Both PKL1-1 mutant Arabidopsis seeds and wild-type Arabidopsis seeds were prepared with bleach solution and washed with sterile water, and all of the plates were taken into the tissue culture laboratory. 5 wild-type seeds were planted onto 5 of the normal agar petri dishes, and 5 mutant seeds were planted on each of the remaining petri dishes. The plates were parafilmed and placed under the plant lights. After 4 days, the plates were placed vertically in Tupperware containers sectioned with lab tape. One week after planting, the root lengths and shoot lengths of each plant were measured for a total of four days using a metric ruler, precise to the nearest millimeter. Statistical analysis was conducted to determine whether or not the gibberellic acid treatment was able to enhance the growth of the mutants. The independent variables for part 1 of the experiment were the concentrations of gibberellic acid and the method of gibberellic acid exposure (directly into the growth medium or applying onto the developing plant). For part 2, the independent variable was the gibberellic acid exposure. The dependent variables were shoot growth, root growth, and any qualitative observations that represented the development of the plant. For both parts of the experiment, the plants not treated with gibberellic acid represented the control. Temperature, light exposure, and method of measuring were some constants for the experiment. For each treatment group, there were five plates with five plants per plate, resulting in 25 trials. Results:In order to determine the most effective method of gibberellic acid application, it was first necessary to calculate the average shoot growth and root growth of the plants over the four-day period. For each of the six treatment groups (high, medium, and low concentrations for both the spray and the infused agar) and the control there were 25 plants that were averaged. The standard deviations of the means were calculated as well, to determine the spread of the data. The average growth of the shoots including the standard deviation is displayed in Table 1.1. In Table 1.1, all of the data has been rounded to the nearest hundredths place, to allow for standardized precision. The days indicate the number of days that the data was collected post- planting. Simply by looking at the data, it is apparent that the plants grown in low and medium concentration gibberellic acid infused agar had the greatest amount of growth. Graphically, the data from Table 1.1 has been displayed in Graphs 1.1 and 1.2. Rather than using one large graph to depict all of the data points, the graphs were divided into two: those plants sprayed with gibberellic acid (Graph 1.1), and those grown in gibberellic acid infused media (Graph 1.2). In both graphs, black bars represent the standard deviations. Graph 1.1Average Shoot Growth for Plants SprayedWith GibberellicAcid Graph 1.1 represents the growth of both the control and of the plants sprayed with gibberellic acid solution. It seems that as the concentrations of gibberellic acid solution increase, so does the shoot growth. All of the standard deviation bars overlap, however, so it is a bit difficult to deduce if this difference is statistically significant. All of the shoot growths were compared to the corresponding day’s control to determine if there was a statistical difference. By the final day, all of the spray treatments were statistically more effective than those control plants not receiving gibberellic acid treatment. To determine this, several t-tests were conducted. A t-test is a form of statistical analysis that is used to determine if the difference between two sets of data is statistically significant. A p value of < 0.05 indicates significance, and is represented by an asterisk on Graph 1.1. Plants Grown in GibberellicAcid Infused Media Graph 1.2 represents the shoot growth of the control and of the plants grown in gibberellic acid infused media. Low and medium concentrations have the largest shoot growth, and looking at the standard deviation bars, they seem to be consistent. Additional t-tests were conducted to compare the treatments to the controls, and on all days other than day 8, all of the shoots of the plants grown in gibberellic acid infused media were statistically larger than the control plant shoots. In order to determine the most effective treatment, the final average shoot growth for each of the treatment groups was plotted on Graph 1.3. T-tests were conducted to compare all of the different treatment groups’ shoot growths to one another. None of the plants sprayed with gibberellic acid solution had shoot growth that was statistically different from the other sprays. The shoot growth of plants grown in low and medium concentrations of gibberellic acid were not statistically different from each other, but did result in statistically greater growth than the high concentration of gibberellic acid infused media and all of the sprays. Final Day (Day 10) Analysis of the root growth in order to determine the most effective gibberellic acid treatment was conducted identically to the analysis of the shoot growth. All of the average root growths are displayed in Table 1.2 along with the corresponding standard deviations. In Table 1.2, low and medium gibberellic acid infused agar have the greatest growth by the final day, followed by the plants sprayed with a high concentration of gibberellic acid. The root growth for plants sprayed with low and medium gibberellic acid seems very similar to the growth of the control. The plants with the least amount of root growth were those grown in high concentration gibberellic acid infused agar. Similar to shoot growth, the data from Table 1.2 is represented graphically in Graphs 1.4 and 1.5. Graph 1.4 depicts the root growth of plants sprayed with gibberellic acid solution, and Graph 1.5 depicts the root growth of plants grown in varying concentrations of gibberellic acid infused media. The standard deviation of each root growth average is shown with a black bar. Graph 1.4Average Root Growth for Plants SprayedWith GibberellicAcid Simply by looking at Graph 1.4, there do not seem to be any major differences between the root growths of the control compared to the root growth of those plants sprayed with gibberellic acid solution. Especially near the earlier days of measurement, the bars appear nearly identical in height. When taking t-tests into account, the high and low sprays resulted in statistically significant root growth enhancement only on day 9. By the final day, the root growth of the plants sprayed with gibberellic acid solution was not statistically different from the root growth of the controls. Graph 1.5Average Root Growth for Plants Grown in GibberellicAcid Infused Media Graph 1.5 reveals that those plants grown in low and medium concentrations of gibberellic acid solution appear to have the greatest root growth.High gibberellic acid infused agar seems to actually inhibit root growth. Using the results from multiple t-tests, it can be concluded that on the final day, all three of the gibberellic acid infused media treatments resulted in root growth statistically different from the control. However, while the low and medium concentrations caused a significant increase in growth, the high concentration caused a significant decrease. In order to determine the most effective treatment, the final average root growth for each of the treatment groups was plotted on Graph 1.6. T-tests were conducted to compare all of the different treatment groups’ shoot growths to one another. Graph 1.6Average Root Growth for Final Day (Day 10) The results for root growth were quite similar to those of shoot growth. Low and medium gibberellic acid infused agar were the most effective treatments, because only these plants were statistically different from the control by the last day of measurement (Graph 1.5). The plants grown in high gibberellic acid infused media were also statistically different from the control, but with a decreased amount of growth. In fact, Graph 1.6 illustrates that the root growth for plants grown in the high gibberellic acid concentration was statistically different not only from the control’s growth, but also from the root growth of all of the other treatment groups. As for qualitative results from Part 1 of the investigation, the plants treated with gibberellic acid are also a bit of a deeper green in color, in terms of qualitative development. After determining that the low and medium concentrations of gibberellic acid infused agar were most effective at increasing root and shoot growth, this treatment was applied to PKL1-1 mutant plants in Part 2 of the investigation. For Part 2 of the experiment, the statistical analysis conducted followed the same basic method as that of Part 1. Averages and standard deviations were calculated for both the root and shoot growths, and plotted on bar graphs. Many t-tests were conducted as well. For shoot growth, the numerical data is displayed in Table 2.1, and graphically in Graph 2.1. As with the data from Part 1, all of the data in Table 2.1 has been rounded to the hundredths place for standardized precision, and the days refer to the number of days post-planting. Looking at the data, all of the shoot growth measurements seem very similar, other than the mutant seeds grown on normal agar (with no gibberellic acid treatment). Graph 2.1 clearly shows that the shoot growth of all the treatment groups, other than that of the mutant seeds planted on normal agar, are very similar. Even the standard deviation bars are very similar.When taking t-tests into account, the mutant seeds planted on normal agar are statistically different from all other treatment groups on each day of data collection. None of the other treatment groups’ shoot growths were statistically different from one another. The average growth for the roots of the plants is displayed in Table 2.2, and it is shown graphically on Graph 2.2. Table 2.2 suggests that the root growth may not have followed the same pattern as the shoot growth for the mutant trials. The root lengths for those plants treated with gibberellic acid have actually grown larger than the untreated wild-type plants. Graph 2.2Average Root Growth For MutantTrials Graph 2.2 visually depicts the increase in growth that the roots of those plants treated with gibberellic acid have had compared to the roots of both the wild-type plants and the mutant plants grown on normal agar. The standard deviations for the plants treated with gibberellic acid are quite large in comparison to the other groups. Similar to the data for root growth, the mutant plants grown on normal agar are had statistically less root growth than that of all other treatment groups on every day of data collection. Additional t-tests also suggest that by the final day of measurement, not only did the plants grown in gibberellic acid have an increase in root growth to allow them to be statistically different from the root growth of the mutants not treated with gibberellic acid, but are also statistically greater than the growth of the wild-type plants. This indicates that the gibberellic acid was fully able to reverse the effects of this mutation on root growth, and even produce plants whose roots are statistically longer than those of non-mutated plants. As for qualitative analysis of the development of the plants, the color of the plants as well as the appearance of lateral roots was observed. The gibberellic acid did not appear to have a large effect on the coloring of the wild-type plants, however it did cause the mutant plants to become a bit of a deeper shade of green. The gibberellic acid did not appear to have a large effect on the amount of roots of each plant. The mutant plants had more lateral roots than the wild-type plants, but this was true even for the mutants not treated with gibberellic acid. Discussion: After completing all of the data analysis, the results from this investigation allowed me to determine both the most effective method of applying gibberellic acid to Arabidopsis plants, and to determine the effect of the hormone on the PKL1-1 mutants. For the first part of the experiment, those plants grown in low and medium concentrations of gibberellic acid had the greatest amount of statistically significant growth. In terms of shoot growth, all of the Arabidopsis plants treated with gibberellic acid had statistically greater amounts of growth than the control plants, as shown in Table 1.1. This is consistent with the findings of Lovell and Booth, who also determined that gibberellic acid positively affects the shoot growth of plants (Lovell & Booth, 1967). As for the roots, I was surprised to find that gibberellic acid had a positive effect on the root growth of the plants for many of the treatment groups, because gibberellic acid is typically thought of as only affecting the shoots of plants. Thus, I rejected the aspect of my hypothesis in which I proposed that gibberellic acid would not impact the plants’ root growth. A potential explanation for this data was that since gibberellic acid causes an increased speed of germination (Gupta & Chakrabarty, 2013), the plants treated with gibberellic acid might simply have been further along in development than the wild-type plants, so their roots were longer. Regardless, for both root and shoot growth, the plants grown in low or medium concentrations of gibberellic acid infused agar had the greatest increase in growth. Initially, I hypothesized that the plants sprayed with gibberellic acid would have more growth than the plants grown in gibberellic acid infused media, but this was not the case, as evidenced by Graph 1.3. I noticed that while applying the spray to the plants, the force of the solution being applied caused the plants to wilt a bit. This may account for the results. Perhaps as an extension to the experiment, it would be interesting to retry the procedure at a later point of development, in which the plants would be more stable. I also hypothesized that higher concentrations of the hormone would have a greater positive effect on the growth of the plants, but for the plants grown on gibberellic acid infused agar, this was not the case. While initially I had assumed that by increasing the concentration there would be increased growth, the data suggested that in Arabidopsis plants, there is an optimal range for gibberellic acid application, which is between 50 and 100 mg/L. At concentrations above this optimal range, gibberellic acid can actually cause the plants to have a significant decrease in growth, as shown in Table 1.1. This was suggested by the data from the gibberellic acid agar infusion trials, as there was no significant difference between the growths of plants sprayed with different concentrations of aqueous solutions of gibberellic acid. Using this data, I have determined an optimal range, but as an extension, it may be interesting to test more concentrations and determine the exact optimal concentration. When beginning to conduct part 2 of the experiment, I decided to only use the most effective gibberellic acid treatment from part 1. By doing so, I would ensure the greatest likelihood of the gibberellic acid having an effect on the mutants. In part 1, though, I discovered that there was not only one treatment that was the most successful; the plants grown on both low and medium concentrations of gibberellic acid infused agar were equally effective, so I chose to use both. For this portion of the experiment, I hypothesized that applying the gibberellic acid would enhance the growth of the mutants, and my data supported the hypothesis. For both shoot growth and root growth, the plants treated with gibberellic acid grew statistically greater than those mutants not treated with gibberellic acid, as evidenced by Tables 2.1 and 2.2. In fact, the root growths for the plants treated with gibberellic acid were also statistically greater than the root growth of the normal wild-type plants. In previous research conducted by the creators of this mutant, gibberellic acid application only partially rescued the shoot growth of the mutants (Henderson et al., 2004). These scientists applied the gibberellic acid exogenously, likely through a spray. By using my method of putting the proper concentration of the hormone into the growth media, the height was fully rescued, and even slightly increased. Using my data, I attempted to develop a proposed model of how the method of gibberellic acid application reversed the effects of the PKL1-1 mutation. In a wild-type PKL plant, the PKL gene works to repress embryonic phenotypes. Essentially, through coding for a CDH3 chromatin-remodeling factor, PKL allows the plant to transition from an embryonic state into an adult state. It reduces the expression of of embryonic phenotypes, such as short stature and many small roots (Henderson, et. al.). In the mutant, this gene is not functional, and thus the embryonic phenotype can’t be repressed. When exogenous gibberellic acid is applied to the mutant, it begins to activate an alternate pathway, in which other CDH genes are activated. This pathway allows the effects of the mutation to be partially rescued. However, the creators of the mutant found that gibberellic acid could not fully increase the short stature of the plant. Using the method that I developed in my investigation (50-100 mg/L gibberellic acid infused into the growth media), the height was fully rescued, and root growth even slightly exceeded that of the wild-type plants. This method of gibberellic acid application was likely successful in activating this alternate pathway. This model is shown below in Figure 3. Although the experimentation ran quite smoothly, there were a few errors that could be corrected for future trials. It was a bit difficult to accurately measure the shoot lengths of the plants, because often times the shoots became twirled and compacted. I did my best, and rotated my ruler to account for all of the different curves of the shoots, but perhaps an easier method would have been to remove the plant from the agar and measure it on a flat surface. If I did this, though, then I could not replant it; thus I could only do this for the last day of measuring. Another small problem that arose was the appearance of mold or bacterial colonies on the petri dishes. Wherever I saw contamination, I quickly removed it with a flame-sterilized scalpel. I also made extra plates, so if I noticed contamination at the start of the experiment, I simply disposed of the plate and used an extra. Unfortunately, there is no surefire way to prevent contamination, but for next time, I would most likely make even more extra plates to have as reserves. The results of this experiment could prove very advantageous to the floriculture and agriculture industries. Modern agriculturists are constantly looking for methods of increasing the growth of plants, but it has become quite controversial to use inorganic materials in order to do so. Gibberellic acid is a relatively easily obtainable organic compound that is already being used in some areas. This research suggests that by using a 50 mg/L to 100 mg/L concentration of the hormone applied directly into agar, plants will have increased amounts of growth. Within the floriculture industry, plants such as orchids are often cultured using agar, so simply infusing a set concentration of gibberellic acid into this media would be a simple procedure. Future research can be done to see the effects of this application method for other growth media, such as soil. This investigation also opens the door to further research with plant hormones such as auxins or cytokinins, and seeing how different methods of application impact their effect on plants. Not only do the results of this investigation apply to agriculture, but they can also have immense benefit in other areas of biotechnology. The study suggests that the method of application of hormones plays just as significant a role as the hormone itself, and sometimes, a new method of application can trigger an entirely new pathway. It would be interesting to attempt to alter methods of application of hormones given to animals or even humans, and see how this impacts their efficacy. Conclusion:
|
What are the effects of gibberellic acid?
Postagens relacionadas
direito autoral © 2024 aprendervalor Inc.
