Determining the Metabolism of GA3 in Four Genotypes of Brassica rapa Anthonia George Biology 240W Section 5 February 13, 2013 ABSTRACT The objective of this experiment was to determine how the different genotypes of Brassica rapa differ in their GA3 metabolism; the different genotypes being, Wild type, petite, elongate and rosette, (the wild type and petite versions have the normal amount of GA3, while the elongate over produces it, and the rosette under produces it). GA3 promotes increased growth rate in plants and internode elongation. Two other treatments were used, water (being the control) and Cycocel which limits the effect of GA3. The experiment was placed in an apparatus with a wicking system which keeps the plants moistened and the plants were kept under 24hr illumination. Initial heights were recorded on the first day and final measurements on the last day. In between this time, the plant had been treated and measured every day around the same time for a whole week (excluding the weekend), using a ruler, spray bottles containing the treatment and tubes. The class data was averaged. At the end of the experiment, the results showed that the genotypes with abnormal amounts of GA3 (elongate and rosette) had the most reaction to it. The results also showed that there was considerable growth in the GA3 treated plants, and an insignificant amount of growth in the Cycocel treated plants since cycocel as described before inhibits GA3 metabolism. The control grew, but not nearly as much as the Gibberellic Acid treated plants, showing that GA3 was functioning. INTRODUCTION According to Campbell Biology 9th edition (Reese et al.,2011), a hormone is a signaling molecule that is produced in tiny amounts by one part of an organism’s body and transported to other parts, where it binds to a specific receptor and triggers responses in target cells and tissues. Plant hormones help coordinate growth, development and responses to stimuli. They are also active at very low concentrations. The major classes of plant hormones are abscisic acid, auxin, cytokinin, gibberellic acid, ethylene, and brassinosteroids (McKeon and Warren, 2013). Gibberellin (which is used in this experiment), also called GA3 promotes growth, by breaking seed dormancy resulting in germination and elongation of internodes. GA3 also stimulates pollen development, pollen tube and fruit growth, regulates sex determination and the transition from juvenile to adult phase. The meristems of apical buds and roots, young leaves and developing seeds are the primary sites for the production of GA3. Gibberellic Acid is responsible for the elongation of stems and internodes in plants (Figure 1). Figure 1- Chemical structure of GA3. The plant used in this experiment is called Brassica rapa or the Wisconsin fast plant (Figure 2). Figure 2- Brassica rapa Wisconsin Fast plants were developed by Dr. Paul William at the University of Wisconsin, and have been bred by him for over 30 years now. Fast plants are a rapid-cycling member of the mustard and cabbage family known as Cruciferae. The other members include Chinese cabbage, canola, and turnips. (McKeon and Warren, 2013). These plants mature and reproduce quickly (have a rapid life cycle), they also have a wide variety of recognized phenotypes and they provide quick results. This plant is ideal for this experiment due to these advantageous qualities. The genotypes of Brassica rapa used were wild type (Wt), rosette (Rs) – dwarf mutant, petite (Pt) – dwarf mutant, and elongate (El) – tall mutant. Three treatments are used on the plants, water, GA3 (described above) and cycocel. Cycocel is among the most reliable and widely used plant growth regulators on the market today. It is a chemical inhibitor of endogenous gibberellic acid synthesis. The overall purpose of this experiment was to determine how these different genotypes of Brassica rapa differ in their GA3 metabolism. This showed the difference in how the hormone works in the different genotypes of the plant. It was predicted that the Gibberellin treated plants would be much taller than the other samples (would have long thin stems) and Cycocel treated plants would have thicker, shorter stems, since it inhibits stem elongation. Of the Gibberellin treated the plants the elongate genotype was predicted to not grow that much because it is an over producer of GA3 and it already has enough GA3, the dwarf and wild type are regular producers of GA3 and was predicted to grow quite well but not as tremendous as the rosettes that are under producers. MATERIALS AND METHODS Figure 3- Plant system setup Each group in the class was given growing plants of all the four genotypes. These plants were planted in a Styrofoam container that had one plant per square cell, and was on top of a piece of wicking material that led into a water tank underneath the plants. The water that was placed underneath contained copper sulfate, which was added to prevent mold growth on the plants. Looking at Figure 3, the setup of the experiment can be further understood. The WT, PT, EL and RS genotypes were grown in separate sections, and the fourth section remained empty. Three different treatments (water, GA3- 100 parts per million, b-9 solution- 1:100 dilution of cycocel) were administered. On the first day of the experiment, the plants were thinned using forceps, leaving one plant to one cell. Each plant was then measured using a ruler and the results gotten were converted from centimeters to millimeters. The different groups measured the plants different ways even though they were not supposed to, some started measuring from the styrofoam and some directly from the soil, so this could have made results vary. After the plants were measured, they were sprayed using three different spray bottles, each contained a different treatment and three different tubes, each labeled with one treatment. To spray the plants, each plant was covered with a tube and about two squirts from the spray bottle were squirted into the tube. Tubes were used to prevent the treatments from touching the other plants. Another precaution taken was using three different tubes for the different sections so as not to mix treatments. Every day after the first day, except the weekend, for one week, someone from the group went to measure and spray each plant with the treatment needed for it. These plants were kept under 24hr illumination and each groups tray was labeled so as not to them mix up and confuse results. A week after the first day, the final measurements of the plants were taken and all the class data was collected and entered into excel. The averages of the replicates for each genotype and treatment was taken and entered into excel. The average of the lab data was then calculated. Wildtype control GA3 cycocel day 1 20.5 19.4 22 day 2 24.8 28.5 27.5 day 3 30.1 33.6 25.8 day 4 day 5 day 6 38.4 41.1 28 day 7 40.8 47.5 29.6 day 8 46.6 62.3 28.5 Petite control GA3 cycocel day 1 18.7 16.8 24.5 day 2 day 3 day 4 day 5 day 8 24 24.3 25.3 day 6 31.3 30.3 25.2 day 7 24 22.5 24 20 32.7 26.2 37 39.3 28.7 Elongate control GA3 cycocel day 1 43.3 42.3 46.8 day 2 53.7 50.8 62.8 day 3 59.4 62.3 66 day 4 day 6 day 7 71.3 91.2 79.1 day 8 84.5 101 80 Rosette control GA3 cycocel day 1 day 2 2.3 5.7 6.3 day 3 4.6 13.3 7.1 day 4 day 7 7.1 23.9 8.5 day 8 9.3 31.1 10.9 2 5.3 3.3 day 5 68 75.3 75.75 day 5 day 6 6 21.4 8.3 Figure 4- Averaged height data for lab section #5 The overall lab data was then used to create four graphs (average height in mm vs. days), which showed the different growth comparisons. RESULTS AND DISCUSSION Figure 5 – plant set up on last day of experiment Figure 5 above shows the plant set up on the last day. All the plants had grown, some very tall and some not so much. The really tall plants had become droopy looking and they could not support themselves. The following graphs were compiled from the data collected throughout the experiment. Wild Type Growth Comparison Height vs. Time 70 Height (mm) 60 50 40 control 30 GA3 20 cycocel 10 0 day 1 day 2 day 3 day 4 day 5 day 6 day 7 day 8 Time (days) Graph 1- Over the course of the experiment, the growth of the wild type plants treated with GA3 seemed to have surpassed the other ones. The graph also shows that cycocel actually inhibited the growth of plants and even made the plants shorter than those treated with just water (control). Petite Growth Comparison Height vs. Time 50 Height (mm) 40 30 control 20 GA3 10 cycocel 0 day 1 day 2 day 3 day 4 day 5 day 6 day 7 day 8 Time (days) Graph 2- The petite plant did not grow that tall overall, the tallest plant did not even reach 40mm. Although the plants were short, they still followed the same pattern of increased growth in the plants treated with GA3, but in this case this did not start happening till the 7th day. As the plants treated with cycocel decreased in growth rate, the more the plants treated with GA3 increased in growth rate, as time went by. Elongate Growth Comaprison Height vs. Time 120 Height (mm) 100 80 60 control 40 GA3 20 cycocel 0 day 1 day 2 day 3 day 4 day 5 day 6 day 7 day 8 Time (days) Graph 3- This graph shows that the elongate genotype metabolized GA3 the most, because these plants grew really tall, with the tallest being about 100mm. This genotype had the tallest plants. The same trend followed here with plants treated with Cycocel having a decreased growth rate and the plants treated with GA3 having an increased growth rate, as time went by. Rosette Growth Comparison Height vs. time 35 Height (mm) 30 25 20 control 15 GA3 10 cycocel 5 0 day 1 day 2 day 3 day 4 day 5 day 6 day 7 day 8 Time (days) Graph 4- Although the rosette genotype had the absolute shortest plants in the whole experiment, they did metabolize a whole lot of GA3, as there was a drastic change in height between the plants treated with GA3 and the ones not treated. Graph 1, 2, 3 and 4 support the hypothesis stated earlier. Gibberellic acid promotes and increases the rate of growth in plants. Cycocel actually inhibited the growth (shorter stems), but in some genotypes such as the elongate, this effect did not start showing till towards end. The plants treated with the water acted as the control and served as a guide to which plants treated with GA3 and cycocel could be compared too. Another thing noticed was that, even know the Elongates grew the tallest, the rosettes which were much shorter, reacted to GA3 in the highest degree (most change in height), probably because it usually under produces GA3 and now was given a lot of GA3. The Elongates grew tall, but it seemed like at some point they reached a maximum height because they are overproducers and have more than enough GA3. The wild type genotype which is a normal producer of GA3 grew at a regular rate, but the petite plants which are also regular producers did not really metabolize the GA3 because of possible hormonal problems that don’t allow them get very tall, so it didn’t really matter how much GA3 it was going to get, nothing would have changed. Possible sources of error would be the part of the experiment where the plants where measured. The rulers were too big to fit in each cell, so people held the ruler at different angles and from different heights, making it difficult to get an accurate reading. In conclusion, in all of the experiments there was a large amount of growth in the GA3 treated plants, with the rosettes having the greatest growth spurts and the elongate plants growing the tallest. The Cycocel treated plants did not show any great amount of growth, in comparison to the control, in any of the experiments. All plants showed growth though, showing that the overall experiment was a success. It was also noticed that GA3 increases growth in all of the different genotypes of the plant Brassica rapa. LITERATURE CITED CycocelÂ®." OHP, Inc. 2005. Web. 13 Mckeon, Brian Sr., and Warren James Jr. 2013. Plant hormones. Biology 240: Form and Function, A laboratory Manual (Penn State, The Behrend College) Reece, Jane B., and Lisa A.. Urry. "Plant Responses to Internal and External Signals." Campbell Biology. San Francisco, CA: Benjamin Cummings, 2011. 824-27. Print. Reynolds, Laura. "Facts About the Wisconsin Fast Plant." GardenGuides. Demand Media, 1997. Web. 13 Feb. 2013. Williams, Coe. "The Story of Fast Plants." Wisconsin Fast PlantsÂ® Program. University of Wisconsin- Madison, n.d. Web. 13 Feb. 2013. Feb. 2013.