Zachary Allbritton
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Experimenting on Mutant Plants
Introduction
Many botanists around the nation experiment on plants to observe the effects of certain changes. Some of these changes can include climate change, relocation, change of light wavelength and/or intensity, and genetic modification. They observe these effects to determine whether or not the tests increase or decrease the amount of produce farmers can produce. In order to achieve the desired results in a short period of time, scientists use a specific kind of weed, called Arabidopsis thaliana. This plant is a small, fast-growing plant that is simple to manipulate and run tests on. Its size makes it easy to observe in labs, and structure allows scientists to get a good idea of whether or not their tests are actually having an effect.
For this lab, we would be imitating those scientists in that we would be using Arabidopsis thaliana to observe the effects of different gene manipulations. Certain genes are turned off, and others are turned on. In most cases, it is only one gene that is modified, so it is simple to discern the function of the modified gene. Each group was assigned a mutant plant, and tasked with the objective of discerning a gene's function, and observing its effects of being either turned on, or turned off.
My group was assigned the gene CHS, which is a gene that controls the way that the plant reacts to the environment. CHS stands for Chalcone Synthase, and is a key enzyme of the flavonoid/isoflavonoid biosynthesis pathway, or the plant developmental program. Besides being a part of the plant developmental program, the CHS gene expression is induced in plants under stress conditions such as UV light, bacterial or fungal infection. Expression of CHS causes accumulation of flavonoid and isoflavonoid phytolexins and is involved in the salicylic acid defense pathway.
CHS is a member of the plant polyketide synthase superfamily. CHS, the most well known representatives of this family, provide the starting materials for a divers set of matabolites (flavonoids) which have different and important roles in flowering plants, such as providing floral pigments, antibiotics, UV protectants and insect repellents.
Our experiment revolves around how both mutant plants, which have the CHS gene turned on, and wild type plants, which have not been altered, react to a stress condition (high-intensity UV light). Four pots containing Arabidopsis will be used (two will hold the mutant plants, two the wild type). One of each plant will be placed under the UV light, and the other two will be exposed to normal daylight conditions. We predicted that the mutant plants would survive better under the stress conditions than the unchanged wild type plant.
Materials
Wild Type seeds
Mutant seeds
Water
4 small pots 2"
Soil
Fertilizer
Regular Light
Ultraviolet Light
Large trays with holes (shared with other peers)
Methods
First, 30 wild-type seeds and 30 Pap-1D seeds were put into two 2ML microtubes, respectively. We then took the seeds and placed them into the refrigerator for 3 days. We then used pipettes to plant 15 seeds from each microtube into the 4 pots. 2 pots had wild-type seeds, One pot held the control seeds, the other would hold the seeds we would conduct the experiment on.. We also did this with the mutant CHS seeds.Once all four pots were seeded, we continued by placing them under a normal fluorescent light. The first 3 days after planting we put a humidity dome over the plants to keep in moisture. After about fifteen days, we began treatment. During the sprouting and experiment phase, we recorded the plants' measurements and made observations every day of the week. We observed bolt height, color, rosette diameter, and size.
Results
Day 1: First Sprouts
This day was the first day we began seeing signs of plant growth. The first few plants are mostly growing in the wild type pots.
Day 6: All Sprouted
All plants have sprouted in both pots, but wild type plants are ahead in the growth process whereas mutant plants are all in the first sprouting stages. First signs of rosettes are growing.
Day 7: Minor Growth
Plants continue to grow, wild type plants remain ahead in the growth progress, mutant plants have very little changes.
Day 8: Slight Growth
No major changes, rosette diameter remains around 10 milimeters at the widest point in the WT pots, where the mutants are at 5.milimeters/
Day 9: Slight Growth
Mutant plants are slowly progressing through the growth phase, wild type plants are still slightly ahead.
Day 10: Slight Growth
Growth continues, wild type plants seem to be about twice the size of the mutant plants in width, around the same in height. Rosetes are beginning to show more on all plants.
Day 13: Major Growth
Plants doubled in size, rosettes now fully visible on every plant in each pot. Height grew slightly as well, but the main change was the diameter of the rosettes' diameter. Wild types are still twice the width of mutants.
Day 14: Major Growth
Plants went through another major growth spurt, not as great as the previous, however. Perhaps increasing their diameter by a quarter the previous day's diameter. Mutant plants' growth might be stunted by gene.
Day 15: Minor Growth, Begin Experimentation
Plants slightly grew, wild type's growth seems to be slowing, and mutants continue to grow. This day will be when we began our experiment. After the initial measurements were taken, we put the pots labeled "Experimental" under high-intensity UV light, and the pots labeled "Control" under the normal daylight situations.
Day 16: Minor Growth
No changes based on experimentation, plants continue to grow. Only differences so far remain that the wild type plants are significantly larger than the mutants.
Day 17: Significant Growth
Day 20; Minor Growth
Bolts are beginning to grow in the center of the rosettes of mutant experimental and control. Only about 2-3 mm tall. No other major changes.
Mutant plants seem to have finally caught up to the wild type plants in their growth phase. Rosette diameters are close together, with mutant plants 10-20 mm thinner than the wild type plants. Rosettes are filling out, and differences in the leaves are starting to become visible. In mutant plants, there is a lighter green edging the rosette leaves, and the leaves are flatter than those of the wild type's, who have a bowl-like shape to the leaves.
Day 22: Minor Growth
Day 23: Minor Growth
No observable changes, other than a slight growth in the plants. Mutant experimental rosette is still mostly smaller than the rest of the plants.
Plants grow at a slightly faster rate, all plants now have a small bolt, the mutant's bolts a few milimeters taller than the rest. Leaves are still retaining their differences. Rosette stem on control WT is longer than experimental rosette stems. Rosette leaves on WT control are thinner than experimental WT.
Day 24: Minor Growth
Final day before Thanksgiving break, this will be the last time we saw before Thanksgiving break. No real changes in plants.
Day 31: Major Growth and Differences
Returned from Thanksgiving break to find that all plants have grown bolts that reach over 100 mm. All except for the mutant experimental, which held an average of 73 mm tall. Siliques are spread along most bolt lengths, but on the mutant experimental, the siliques are clustered towards the top, by the flowers and most of the leaves. Possibility that this could be the CHS gene manipulating how the plant is reacting to the new environment.
Day 33: Minor Changes and Issues
While plants continued to survive, some of the plants' rosettes began to die out. The mutant experimental's rosettes had almost half of their leaves dead, and the others' were starting to die. Most likely caused by lack of water. Though plants were watered through Thanksgiving break and previous days.
Day 35: Final Day
The experiments' final day came through, with some plants still surviving. Most of the experimental mutants' rosettes are dead, but the bolts are stil lgreen and surviving. The experimental control is mostly dead as well, with the only main surviving plants being the two control plants.
The wild type experiement began shrinking a day or two before the mutant experimental plant, and the plants' death was brought upon by the lack of water, and by the time we returned to attempt to save the plants, it was too late. The mutant, while it lived, seemed to flourish better under a stress condition than the wild type plant, allowing the plant to survive better in a different environment than the plant was used to.
The mutant CHS was smaller in all ways than the wild type plant. Rosette diameter was smaller and the bolt height was nearly a quarter of the wild type’s. The mutant’s growth seemed to be stunted by the UV light, and the siliques were clustered towards the top of the bolts, rather than spread out along the length of the bolt. The mutant also doesn’t seem to be growing a second bolt, while the wild type is. This is a sign that the plant is reacting differently to its environment and that the CHS gene is being expressed.
The mutant exposed to the stress condition of UV light was significantly smaller, and had a much shorter bolt than that of the mutant control. The siliques along the bolts were clustered towards the flowers at the top of the bolts for the experimental plant, whereas the control had them spread all along the length of the bolt
In conclusion, the mutant plants survived significantly better than the mutant plants, and we would have aquired better results if our plants had not died early. The mutant expirimental plant was the smallest of the four pots, which could be a sign of the CHS gene changing how the plant is reacting the new environment. Since the plant partially grew in normal daylight for its first stages, the CHS gene had to readjust to a new situation that the environment put forth.
The effect of the gene mutation showed to be quite significant. Although it took the mutant longer to sprout, which must be taken into consideration, the overall growth rate of the mutated plant was much slower than that of the wild. Through most of the plants' growth, the wild type was double the size of the mutant, and stayed that way until we returned from Thanksgiving break. Along with the growth difference we noticed that the plants phenotypes were slightly different from each other. The Wild Type's leaves had a fuzzy texture to it, and the mutant's was smoother. The wild type's rosette also seemed to grow thicker, with wider leaves that were also a slightly darker shade of green when compared to the mutant. The experimental mutant also seemed to have a much smaller bolt than the rest by about half. The siliques were all clustered towards the flowering end of the bolt, and stood straight where the others bent over themselves. Over the course of the experiment, the wild type experiment pot began dying a few days before the lack of water took effect on all the rest of the plants, and the mutants survived the best of all the plants. Overall, we noticed that there was a difference between the two plants, showing that under normal conditions the Mutated Type is already significantly smaller than the Wild Type, and better suited to adapting to the environment.
If we were to do the experiment again, there woudl be a few factors I would incorporate differently. The first of which being a watering schedule. If we knew when the plants were last watered, and when the plants had to be watered, it would allow the plants to live on, uninhibited by a lack of water, and allowing us to see if it really is the gene at work, or the environmental factors. Another thing that I would have changes is the way all the plants are put under light. We used a metal shelf with flourescent lights to grow the plants in. We had the different kinds of light underneath the lfourescent, and had confusion among where the plants should be placed. If we seperated out the lights and added a list to the plants that needed to be under which light, that confusion could be solved.
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