The foundation of genetics lies with the principles that Gregor Mendel outlined after his experiments with pea plants where he discovered the relationship between physical characteristics, or phenotype, and genetic traits, or genotype. This experiment aimed to reproduce Mendel’s results with the Brassica rapa plant, noted for it’s fast generation time, and anthocyanin, a purple pigment that can be visually tracked through subsequent generations. It is important for experiments resulting in scientific discovery to be replicable and peer reviewed.
Since Mendelian genetics are the foundation of scientific education, including answering questions about evolution and heredity of beneficial or fatal genes, his experiment is a valid and important choice. The hypothesis was that the presence of anthocyanin in Brassica rapa follows Mendel’s laws. By germinating the P1 generation and creating the F1 and F2 generations through pollination the hypothesis was tested. The experimental hypothesis was accepted with error and the null hypothesis that these results were due to chance was rejected.
A phenotype provides a clear visual cue for examining the inheritance pattern of genotypes and whether or not these patterns follow Mendelian genetic principles. Gregor Mendel is someone who is often referred to as the “father of genetics,” and discovered important theory pertaining to heredity. He did this by studying traits of pea plants in the garden of a monastery and found two main observations to be supported by the collected data from his experiments about the inheritance of traits in pea plants. As a result of Mendel’s experiments with pea plants, he developed two observations, which together comprise Mendelian genetics. The first observation is The Law of Segregation, which states that paired alleles separate during the formation of gametes.
The second observation is The Law of Independent Assortment, which states that each allele segregates independently of the other allele in gamete formation (Pearson 1904). This experiment with Brassica rapa aims to replicate Mendel’s results with the pea plants leading to his conclusions about genetics that currently provide the foundations for genetic theory. Brassica rapa is a plant that has a comparatively short, natural (non-induced) growth cycle from seed to reproductive maturity than most other plant species. This makes Brassica rapa, also known as “fast plant”, an ideal choice for many scientific applications, from basic reproductive biology to university level studies. Anthocyanin, a pigment found in plants, results in a purple stem phenotype for the Brassica rapa plant (Gould, 2009). A phenotype is the observable characteristic(s) in an individual (Rollo, 1994). Genotypes are the genetic makeup of the individual plant or animal.
The interactions between genotypes when animals or plants breed results in a new generation with a unique phenotype (Rollo, 1994). For example, the observable purple color of the stem of the Brassica rapa plant is the phenotype of a gene. A gene is a small part of the DNA strand that tells the cells that make up the body of the organism what to do. Genotypes are a code that can help explain why genes can be expressed, or not expressed. The genotype of Anthocyanin is as follows; (ANL/ANL) wild type, (ANL/anl) (both result in purple stems), or (anl/anl) which is a recessive form resulting in green stems. Anthocyanin is dominant, and can be either homozygous dominant (the genotype code with two capital letters) or heterozygous dominant (the genotype with one capital letter code and one lowercase code) (Gould, 2009).
Dominant genotypes mean that only one of the genotype codes in capital letters needs to be present for the phenotype to be activated. Recessive genotypes mean that two lowercase genotype codes have to be present for the phenotype to be expressed. Another examples of a gene found in the Brassica rapa plant include the yellow-green gene (ygr), which is homozygous recessive, needing both lowercase genotypes to be expressed. Other forms of the genotype, (ygr/ YGR) and (YGR/YGR) will result in green leaves. A third gene in Brassica rapa is the rosette mutant, homozygous recessive. The genotype needed for the short, rosette plant form is (ros/ros). The other two genotypes (ros/ROS) and wild type (ROS/ROS) will result in the normal form of the plant. The phenotypes and genotypes are related in that the phenotypes provide a visible indication of the genotype. This is true in an individual with a homozygous recessive gene.
However, in the case of dominant genes, since only one copy is needed for the phenotype to be present, then the second copy is not indicated. The second copy can be identified process where two individuals (P1 and P2) with the same dominant phenotype, called the parental generation, are bred. This produces an F1 or first generation of offspring. The F1 generation can also be bred and produce an F2 generation. Each individual in the F1 and F2 generations receives one copy from each parent of the 3-letter genotype code, called an allele. Gregor Mendel theorized that certain combinations of alleles in a genotype would result in a specific ratio of phenotypes expressed in each generation. For example, in the case of the dominant heterozygous anthocyanin genotype, the P1 with (ANL/anl) crossed with the P2 (ANL/anl) would result in a 1:2:1 ratio for genotypes (ANL/ANL), (ANL/anl) and (anl/anl).
However, since the phenotype is dominant and only one copy is needed, the phenotypic ratio would be 3:1 (purple stems: green stems). For Mendel’s genetics to work two things must be true. The Law of Segregation or “The First Law”, states that paired alleles separate during the formation of the gametes. Gametes in for the Brassica rapa are the seeds the plant produces. Gregor Mendel discovered this by observing traits that were inherited in pea plants and noticing that alleles that were originally paired would separate. The Law of Independent Assortment states that each allele segregates independently of the other allele in gamete formation (Pearson, 1904)
This is important for education regarding inheritance and genetics because it is one of the foundation principles concerning heredity. Heredity and genetics provide the basis for answers to questions about evolution of species, presence of disease and genetic faults or other traits of interest in familial lines. All of these questions of substantial significance have answers that are based in Mendelian genetics. In response to the question: does the inheritance pattern of anthocyanin in Brassica rapa follow Mendelian laws; the hypothesis was formed that the presence of anthocyanin in Brassica rapa follows Mendel’s laws. If anthocyanin follows Mendelian genetics, then the F2 generation will exhibit the 3:1 phenotypic ratio.
Materials and Methods
Plant Data (Table 1)
Day 112 seeds planted
Day 8All plants with green stems removed
Day 22First cross pollination of plants
Day 24Second cross pollination of plants
Day 31Plants stopped being watered
Day 38Pods removed from plants and seeds removed from pods
Day 43Seeds re-planted
Table 1 is actions taken with the seeds and plants throughout the plant life cycle.
In order to test Mendelian genetics and determine how dominant and recessive alleles are inherited in Brassica rapa twelve seeds were planted. The seeds were Wisconsin fast plants from Carolina Biological Supply, genotype anl/anl and ANL/ANL, generation F1. The seeds were planted in a six-compartment seedling tray on day one of the experiment. Ordinary potting soil was used to plant the seeds and each compartment was filled seventy to eighty percent full of soil. Two seeds were then planted diagonally from each other in each of the six compartments. A small pellet of Miracle Gro shake ‘n feed fertilizer was placed in the middle of each of the six compartments, having been mindful of not placing the fertilizer to close to either of the seeds.
The six compartments were then filled close to the top with soil, another twenty percent. The seedling tray was placed in a water tray and was constantly watered. A LED bank was used as a source of light for the plants, which was on constantly on without being turned off. Then, on day eight of the experiment the plants were checked for any green stems and all plants containing green stems were removed. On day twenty-two the first cross pollination of the plants was performed by using a small paintbrush to gather the pollen and then brushing the other flowers on the plants in the surrounding five plants with the same brush. This same procedure was done to all six plants. The plants were then placed back in the water tray and under the LED bank.
On day twenty-four the second cross-pollination was performed with same procedure as in the first cross-pollination. On day thirty-one the plants were removed from the water tray so that the seedpods could dry out. On day thirty-eight the pods were removed from the plants and the seeds were removed from the pods. The seeds were then counted and put in small yellow envelopes to dry out for re-planting. On day forty-three the seeds were re-planted on a paper towel with no soil. The seeds were placed on a 1-1.5cm grid in a large black tub a covered with a lid. The container/tub was placed in the windowsill where the plants received sunlight and are not in a LED bank. The seeds were sprayed with tap water everyday. This experiment was performed mid summer
Image 1 shows the growth of the Brassica rapa plant (Wisconsin Alumni Research, 2014)
Table 2 compares the difference in the visual confirmation of the anthocyanin genotype
In order to confirm the presence of anthocyanin genotype in the Brassica rapa plant, the stems of the plant were examined for the presence of purple pigment. The purple pigment is a unique phenotype that is evident as positive confirmation of the anthocyanin genotype. The images in Table 2 show the physical differences between the negative anthocyanin genotype resulting in a green-stemmed phenotype and the positive anthocyanin genotype resulting in a purple-stemmed phenotype.
Image 2 shows an example Chi square result of the genotypes and phenotypes of an F2 generation with the 3:1 (dominant: recessive) ratio for green and yellow seeded plants. Green is dominant with seventy-five percent of the generation while yellow is recessive with twenty-five percent. In Medelian genetics, under the rule of “The First Law” or the Law of Segregation if the F1 generation is homozygous dominant and homozygous recessive then the ratio of the F2 generation will be 3:1 dominant to recessive. Therefore, there will be seventy-five percent purple-stemmed plants and twenty-five percent green-stemmed plants.
Section 001 Plant Results (Table 3)
Table 3 shows the difference between the expected stem colors predicted by vs. resulting colors and the standard deviation difference.
This table compares the expected and observed values of the plants through finding the degrees of freedom. For this experiment, the null hypothesis is excepted when there is less than a five percent margin of error, or finding a number on the Chis-Square distribution table that is higher than 3.84. To find this number the observed values for both the green and purple-stemmed plants are summed together. To find seventy-five percent of the summed number to determine the amount of expected purple-stemmed plants the sum of the observed values was multiplied by .75. I also multiplied the sum of observed green and purple plants by .25 to find twenty-five percent of that number, or the number of expected green-stemmed plants (119.75).
Then to find the deviation I subtracted the expected values for both the green and purple-stemmed plants from the observed values (19.25). To find the denotation, the amount of the deviation was squared; this produced a denotation of 370.5625. The final calculations were made then by dividing the denotation for both the green-stemmed and purple-stemmed plants by their expected values, this produced the results 1.0315 for the green-stemmed plants and 3.0945 for the purple-stemmed plants. To find whether the null hypothesis will be supported or rejected these two numbers are added together. When these two numbers are added, a result of 4.13 is produced. Using this number Table 4 can be referenced. When Table 4 is referenced 4.13 is higher than 3.84, which rejects the null hypothesis and supports the experimental hypothesis.
Section 001 and Section 002 Plant Results (Table 4)
In table four similar procedures were followed to find the degrees of freedom and support or reject the null hypothesis. The first step of the procedure was to add the observed values for the purple-stemmed plants of both sections and the observed values of green-stemmed plants of both section; the results were 745 purple-stemmed plants and 300 green-stemmed plants. These two numbers are summed to produce a resulting value of 1045. To find the expected values .75 and .25 to find the expected values of purple and green-stemmed plants then multiplies the sum of the observed values. Once the expected values have been found the first deviation can be determined by subtracting the expected values from the observed values.
Table 5 is the distribution value for a chi square
Brassica rapa plants were grown over a period of forty-three days and observed for the anthocyanin genotype resulting in the purple pigment of the stem phenotype in order to observe Mendelian genetics. The results of this experiment confirm the hypothesis that the presence of anthocyanin in Brassica rapa follows Mendel’s laws. A total of four hundred and seventy nine plants were observed for this period. Out of those, three hundred and forty were confirmed to have the purple stems representing a positive conformation of the expression of the anthocyanin genotype. The remainder, a total of one hundred and thirty nine plants were observed to have green stems, which were negative for the expression or presence of anthocyanin.
The expected results were three hundred and fifty nine plants positive for the expression and presence of the anthocyanin, and one hundred and twenty Brassica rapa plants. Examining the results from tables three and four the results of table three for section 001 show a deviation of 4.13, when there is only 1 degree of freedom. In order for the null hypothesis to be rejected this number would need to be less than 3.84, to show that the results of the experiment were due to chance. In the case of table three the resulting number was greater than 3.84, showing that there is less than a five percent chance that these results were due to chance, rather than error. For section 001 the results supported the experimental hypothesis, producing a ratio of close to 3:1 not based on chance.
For the combination of the data for sections 001 and 002, the null hypothesis is also rejected because the calculations performed produce a resulting deviation of 7.66 which is much higher than 3.44 providing support for the experimental hypothesis as well. When the two sections are combined there is very little probability that the results are due to chance. Possible error exists in multiple variables. Using LED light instead of sunlight could have potentially reduced the photo-synthetically active radiation or PAR available to the plant, which would limit the amount of purple pigment able to be produced through the expression of anthocyanin. Additionally the lack of proper light spectrum could have caused metabolic problems resulting in improper growth and flower production that would have provided incorrect visual cues for the proper stage of growth to observe the anthocyanin expression correctly.
An alternate form of error could have been the micronutrient and macronutrient components of the soil used. A lack or surplus of critical nutrients could have resulted in premature death of the Brassica rapa plant resulting in an altered life cycle. Other forms of error include humidity of the air, improper germination techniques, unstable plant genetics, and improper handling techniques during shipping, etc. This experiment can be improved by isolating and controlling for more variables such as the potential forms of error listed above and providing a more rigid experimental environment. LED lighting, for example, vary widely in production and may only use two parts of the PAR spectrum, up to fifteen parts of the spectrum. Temperature is critical to plant growth as evidenced by certain plants growth proximity to the equator, providing different levels of heat during different seasons.
In conclusion, Medelian genetics are a foundational principal of scientific education. Gregor Mendel’s observations have led to the answers of questions regarding evolution of species, heredity of fatal or advantageous genes including environmental influences, and provide a glance into the future of humanity in the biological and technological horizons. Mendel discovered important basic factors regarding heredity, genotype and phenotype. One of these factors or rules is that if the F1 generation contains two parents that are phenotype heterozygous dominant than the F2 will produce a 3:1 phenotypic ratio. In order to test this an experiment was conducted by examining the heredity of anthocyanin in Brassica rapa. Through this experiment the null hypothesis was rejected, supporting the experimental hypothesis that the presence of anthocyanin in Brassica rapa follows Mendel’s laws; and that these results are not due to chance.
One of the most important factors when determining the results from this experiment is Chi-square and understanding how it is affected by Medelian genetics. If the Chi-Square distribution had been less than 5% then the results were due to chance, any variation in results would be due to error after 5% therefor we will accept the experimental hypothesis, the null hypothesis is rejected because chance alone was not responsible for any deviation. This means, through experimentation, that Mendel’s Law of Segregation is supported and the F2 generation will produce a 3:1 ratio. The expected results did not coincide with the observed results; which was determined through the Chi-Square goodness-of-fit test to be due to error, rather than chance. These errors could be a caused by numerous factors, that could be corrected to produce more accurate results.
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