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- 1. Section #6 December 4, 2012 Kendall Marine Anthocyanin: Inheritance Pattern Jecelin Espinal, Brittany Feiten, Emily Hornet, Karina Abstract During this semester in lab, the class conducted an experiment that explored the inheritance of traits in the plant, Brassica Rapa. The Mendelian model aided the class, as we used it in order to develop a hypothesis regarding the patterns of inheritance found in the plant, Brassica Rapa.After conducting the experiment the group came to conclude, that due to the presence of purple pigment (anthocyanin) contrived to be a dominant trait and followed Mendel laws. Overall, the results were inconclusive to the fact that trial and error played a critical role in the experiment. Introduction Gregor Mendel was an Australian monk who discovered the basic ideas of heredity through experiments he conducted in his own garden. Around 1854, Mendel began to conduct experiments about the communication of hereditary traits in plant hybrids. During Mendel¨s time it was universally accepted that a hybrid would return to its original form, and was incapable of creating new forms. Through various year of hard work, Mendel found many new ideas that are now known to be ＾Mendelian Laws￣. One of the laws he discovered was the laws of segregation, this concluded that there are dominant and recessive traits passed on casually from parent to offspring. Another law called, the Law of Independent Assortment, concluded that traits were passed on autonomously of other traits from parent to offspring. Overall, Mendel¨s laws played a huge role not only in genetics but biologically as well. Gregor Mendel¨s laws showed the pattern of inheritance from parent to in and also provided reasons for variations seen in the offspring. Throughout the semester the general 1 bio class conducted an experiment that put Mendelian Genetics to the test. The scientific question asked was, does the inheritance pattern of anthocyanin (purple pigment) follow Mendelian Laws? The group hypothesized that the presence of purple pigment (anthocyanin) is inherited as a dominant trait and does follow Mendelian laws. Before experimenting the class predicted, if anthocyanin is inherited as a dominant trait, then the F2 generation will follow Mendelian genetics resulting in 3:1 phenotypic ratio. In order to carry out the experiment the class used the fast plants which contain strains of Brassica Rapa. According to our lab manual it stated, that the Brassica Rapa have a rapid breeding cycle from seed to seed in 35 days. Due to their rapid breeding cycle, plants in the Brassica family are ideal models for teaching and research. In the experiment conducted the class investigated these traits: presence or absence of anthocyanin, yielding green or purple of purple plants, and color of plant.
- 2. Materials and Methods Before conducting the experiment obtain the following materials in order to complete successful research. Obtain seeds of designated phenotypes, a seed collecting pan, a few small envelopes, wicks, labeling tape, Styrofoam quads, and a florescent light bank. Also, obtain water, a dropper, a potting mix, fertilizer pellets, a watering tray, and a petri dish with filter paper. Once you obtain these materials the experiment can be conducted accordingly. Table 2 Approximate Date Date Activity Initials Day 1 08/21/12 Plant F1 hybrid seeds. J.E. Regularly check water. (See planting seeds) Day 4 or 5 Observe seedlings and J.E. record number of each phenotype in Table 3. Thin plants to one per cell. Days 14,16,20 Pollinate on 3 days; J.E. pollinate at least 6-8 followers. (See pollinating flowers) Days 20 to 39 Remove buds and J.E. shoots. Day 39 Harvest F2 seeds and J.E. germinate in petri dishes. (See Harvesting & Germinating seeds) Day 42 Count and record J.E. number of each phenol type in Table 4. Table above as seen in the lab manual by Dr. Tod Duncan. ＾The Pearson Custom Library for the Biological Sciences. Symbiosis. In order to complete the experiment successfully you need to obtain the following materials from you biology lab instructor. First you will need 12 F1 seeds that resulted from the cross between a homozygous plant that contained anthocyanin and a true- breeding plant that did not contain anthocyanin. Next, obtain 1 quad and other planting supplies. After, get 2 mature plants with parental phenotypes. After all the above is complete, obtain 6 seeds from homozygous green plants, and 6 seeds from the homozygous variegated plants. Then, obtain 2 quads that will be
- 3. used to plant the seeds. After, get 5 inch by 8 inch index cards that will be used later on in the experiment. Finally, obtain a few stakes or small wooden applicator sticks. The following procedure must be followed in order to plant the seeds, pollinate the flowers, and harvest and germinate the seeds of the next generation. In order to plant the seeds correctly they must follow the following. In each cell of the quad add one wick. Make sure the wicks tips extended from the holes in the bottom. Then add plotting soil to each cell of the quad until it was half full. Adding a little more soil and pressing down on the soil with a finger until a depression is present. Then, add two or three seeds to each cell and cover it up with soil. Obtain a dropper and water each cell with the seeds until the water drips from the wick. After, the dripping occurred they placed the quad on a watering tray and placed in under a florescent light bank. Make sure the lights were maintained 2-3 inches above the growing plants and on for 24 hours a day. After, placing it under the light bank, label the quad with their names, the date, and the plant types. After 4 to 5 days they went back to the light bank, and recorded the phenotypes, they removed all but one of the large healthy seedlings in each cell. Next allow two or three flowers to open up on most of the plants. Approximately wait 14 days after the planting of the seeds. She went with her group and they used a ＾bee-stick￣, which was made by gluing a dry honeybee thorax to the top of a toothpick, they transferred pollen from one plant to another one. After, she saved the stick by inserting it into one cell of the quad. She then used the stick to pollinate again 2 to 4 days later. After, the third pollination her group pinched off all the unopened buds. Then, they removed and disregarded of all the new buds and shoots for the following two weeks. After, 21 days after the pollination the seeds were ready to harvest. They removed the quad with the plants from the watering tray and dried it for 5 days. After, she removed all the dry seed pods and rolled them between her hands over the collecting pan to free the seeds from the pod. They took the seeds and stored them in an envelope and labeled it with their names, the date, and the seed type. Following they germinated the seeds by moistening a piece of filter paper in the petri dish, labeling the paper with their name, the date, and the seed type. They made sure they poured off any excess water. Then, they placed 25 of the harvested seeds in neat rows in the upper thirds of the filter paper. She then placed the petri dish tilted on end in a water reservoir and added 2cm of water. The whole class placed the dish and reservoir under the light bank. After 46-98 hours passed, they observed the seedlings and recorded the phenotypes in the appropriate table. The group recorded the results, and performed the chi- square on the class results. Results (Table 4) Team No. Purple Number of Plants No. Green Team Purple Green 1 10 26 7 7 12 2 25 13 8 3 13 42 9 4 10 31 10 5 43 4 11 6 6 3 12 Class total Purple: 130 Green: 165
- 4. The table above shows the totals for the green and purple plants in our class as a whole. Table 5: Chi-square Equations and Results for Section 6 Data Purple-stemmed Plants Green-stemmed Plants Observed (o) 130 165 Expected (e) 221.25 73.75 Deviation (o C e) or d -91.25 91.25 Deviation2 (d2) 8326.56 8326.56 d2/e 37.63 112.9 Chi-square: Χ2=Σ (d2/e) =150.74 Table 6: Chi-square Equations and Results for Total Sample Data Purple-stemmed Plants Green-stemmed Plants Observed (o) 3113 1445 Expected (e) 3418.5 1139.5 Deviation (o C e) or d -305.5 305.5 Deviation2 (d2) 93330.25 93330.25 d2/e 27.3 81.9 Chi-square: Χ2=Σ (d2/e) =109.20 Aggregate Data Table 7 Section # Purple-stemmed Plants Green-stemmed Plants 1 79 28 2 213 101 3 49 61 5 85 33 6 130 165 7 51 22
- 5. 8 8 3 9 181 82 10 154 71 11 83 31 12 62 24 13 104 35 14 1102 468 15 15 18 17 64 53 19 201 33 21 180 71 22 170 73 24 182 73 TOTALS: 3,113 1,445 Conclusion Overall, the results were inconclusive due to many errors that simply did not refute nor support the hypothesis. The hypothesis stated, the presence of purple pigment (anthocyanin) is inherited as a dominant trait and follows Mendelian laws. According to table 4 above, which was the class data, it expressed a ratio of 130:165 green stemmed plants being dominant. According to table 7, which was all the gen bio sections, the purple stemmed plants were dominant with a 3,113:1,445 ratio. So, it seemed as though in the class (section 3) the green-stemmed purple trait was dominant versus the purple, refuting our hypothesis. Also, according to all the section¨s data combined the purple-stemmed trait was dominant, supporting the hypothesis. Then, using the data from tables 6 and 7 it proves that the green-stemmed plants would produce more versus the purple-stemmed plants. Overall, due to the data supporting and refuting the hypothesis, this lead
- 6. to in conclusion. Some errors could be simply to fact that the F1 plants were not pollinated correctly leading to inaccurate crossed between the F1 plants. Also, one of the most common errors occurred was the counting of each phenotype on day 42. This could have led the results in a complete different direction due a simple counting error. The hypothesis was supported by some of the data collected and refuted as well, leaving an inconclusive result. In future replications of this experiment many things can be improved. For example, each class after planting the seeds on the first day should be more actively involved in the growth of the plant and keep a close eye. The class did not really spend much time neither planting nor monitoring the plants which could have led to the neglect to the plants. If future gen bio labs are more actively involved then this could limit the amount of errors, leaving a clear conclusion.