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Plant Dihybrid Report

Diana Miculescu

Georgia State University

April 19, 2015

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Introduction

Through many experiments with pea plants, Gregor Mendel observed patterns in

the inheritance of physical, observable traits. In his experiments, he crossed pure-

breeding parents as well as hybrids and dihybrids. From his results, Mendel observed

3:1 ratios and termed the trait observed in the larger proportion as the dominant

characteristic and the smaller proportion as the recessive characteristic (Yoshio, 2013).

His results led him to propose theories about the method in which the progeny inherits

traits from the parental generation. These theories would later be called the laws of

segregation and independent assortment. The law of segregation states that the

variations of a trait, or alleles, segregate during meiosis so that the offspring obtains a

single allele from each parent (MacLeod, Tweedie, Tait, et al., 2015).

After performing crosses of plants that differed in two traits, called dihybrid

crosses, a modification of the 3:1 ratio was observed. For example, in a dihybrid cross

of a plant the parental generation (P0) contains one parent that is homozygous

dominant for both traits while the other parent is homozygous recessive for both traits

(or one parent homozygous dominant for one trait and recessive for the other). The

cross of the dihybrid leads to a hybrid generation (F1) in which offspring inherit one

dominant allele and one recessive allele for each trait. Once the hybrid generation self-

fertilizes, the offspring (F2 generation) results in a phenotypic ratio of 9:3:3:1. The “9”

part of the ratio refers to the plants that display the dominant phenotypes for both traits.

The “3” part of the ratio refers to the plants that display one of the dominant phenotypes

for one gene and the recessive phenotype for the other gene. Lastly, the “1” part of the

ratio refers to the plants that display both recessive phenotypes for both genes. This
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9:3:3:1 ratio is characteristic of dihybrid crosses. The findings of the dihybrid crosses led

to the proposal that alleles of different characteristics segregate independently which

ensures the transmission of traits to offspring occur independently of one another, now

known as the law of independent assortment (Miko, 2008).

Mendel’s findings in his experiments with pea plants led to the development of

theories of inheritance which would later be combined with the chromosomal theory of

inheritance (Vicedo, 1990). Mendel’s accuracy in his proposals is significant given that

no information about alleles, genes, chromosomes, or genetic material was yet

discovered.

To investigate the laws of segregation and independent assortment and to

observe the Mendelian laws in action, we will use the dihybrid offspring of the rapid-

cycling Brassica rapa that are homozygous for the two traits of stem and leaf color. The

purple color (dominant) in the stem of the plants is caused by anthocyanin which

controlled by the gene anl (Burdzinski & Wendell, 2007). Plants inheriting anl/anl for

stem color will be green (recessive) because the expression of anthocyanin is

suppressed (Burdzinski & Wendell, 2007). The leaf color of light green is controlled by

the gene ygr. Plants inheriting ygr/ygr for leaf color will be light green (recessive), while

those inheriting YGR/ygr or YGY/YGR will be dark green (dominant) in leaf color

(Slankster, Chase, Jones, Wendell, 2012).

In addition to crossing dihybrid plants, we will analyze the resulting progeny

proportions with a chi-square test. This will help determine if the deviations of the

observed count for each phenotype from the expected ratio of a dihybrid cross (9:3:3:1)

are significantly different and if the deviations were due to chance. If the possibility of
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chance occurrence in percentage for 3 degrees of freedom lies above 0.05 (upper-tail),

then there is a high chance that the deviations of observed ratios from expected ratios

are due to chance. However, if under 0.05, then the deviations are significantly different

and there is little chance that the deviations in observed ratios are due to chance.

Methods and Materials

Reservoirs were filled with water, blue algae-control squares were placed in the

water and a saturated water mat was placed on the lid. One wick was placed in each

cell of a quad and each cell was slightly filled with moist soil. Two to three fertilizer

pellets were placed in each cell, more moistened soil was placed in each cell, and then

shallow depressions were made in the soil. Two to three seeds of the offspring (F1

generation: purple stem, dark green leaf anl/ANL, YGR/ygr) of the parent generation (P1

generation: non-purple stem, dark green leaf anl/anl, YGY/YGY and P2 generation:

purple stem, light green leaf ANL/ANL, ygr/ygr) were placed in each depression and soil

was sprinkled on top to cover the seeds. Each cell was watered until the wicks were

saturated. The quads were placed under the lighting system provided in the laboratory.

The plants were watered as needed and the reservoirs were refilled as needed for 2

weeks.

After 2 weeks, pollen was transferred back and forth from the F1 generation

using bee sticks to imitate cross-pollination among the plants.

Twenty days after the cross pollination, the plants were removed from the

reservoirs and allowed to dry out. Once the plants dried out, the seeds (F2 generation)

were harvested from the seeds pods. The harvested seeds were placed on a wet filter
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paper in a petri dish and placed under the lighting system for a week to sprout (until

stem and leaf color were observable).

Results

Of the seeds in the petri dish, the results of the seeds with observable and

distinguishable stem and leaf colors were recorded (regardless if they were alive or

partly dried out). The results observed for Group 1 were 19 plants with purple stems and

dark green leaves, 6 plants with purple stems and light green leaves, 5 plants with non-

purple stems and dark green leaves, and 2 plants with non-purple stems and light green

leaves (Figure 1, Table 1). The class results were combined to yield 26 plants with

purple stems and dark green leaves, 9 plants with purple stems and light green leaves,

14 plants with non-purple stems and dark green leaves, and 4 plants with non-purple

stems and light green leaves (Table 2). The 2 value was calculated to be 2.3793 with 3

degrees of freedom (Table 2).

Figure 1. Brassica rapa (F2 generation) plants pictured after 7

days
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Table 1. Phenotypic and genotypic results of stem and leaf colors of Brassica rapa (F2
generation) from Figure 1
Phenotype purple stems, dark purple stems, light non-purple stems, non-purple stems,
green leaves green leaves dark green leaves light green leaves
Genotype* -/ANL, YGR/- -/ANL, ygr/ygr anl/anl, YGR/- anl/anl, ygr/ygr
Group 1 19 6 5 2
Class combined 26 9 14 4
*(-) in genotype indicates either dominant or recessive allele may be present with no phenotypic
difference

Table 2. Calculation of 2 value using “Class combined” results from Table 1

Phenotype purple stems, dark purple stems, non-purple stems, non-purple stems,
green leaves light green leaves dark green leaves light green leaves
Observed value 26 9 14 4
Expected value* 29.8125 9.9375 9.9375 3.3125
Deviation 3.8125 0.9375 4.0625 0.6875
Deviation squared 14.5351 0.8789 16.5039 0.4726
Deviation squared/
0.4875 0.0884 1.6607 0.1427
expected**
*expected values calculated from observed value of 53 total plants for a phenotypic outcome of 9:3:3:1.
**sum of deviation squared/expected =2= 2.3793
Degrees of freedom= 4-1=3

Discussion and Conclusion

For the resulting total (class combined) of 53 seeds in the F2 generation,

approximately 30 were expected to have purple stems and green leaves, 10 to have

purple steams and light green leaves, 10 to have non-purple stems and dark green

leaves, and 3 to have non-purple stems and light green leaves (Table 2). Of the

observed (class combined), the number of non-purple stems and dark green leaves was

considerably more than expected and of the purple stems and dark green leaves less

plants were observed than expected (Table 2). All plants that were phenotypically non-

purple stemmed must have a genotype of anl/anl. All plants displaying some pigment of

purple must at least one dominant allele of ANL. All plants that were phenotypically light
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green leafed must have a genotype of ygr/ygr, and all plants displaying dark green

leaves must have at least one dominant allele of YGR.

To determine whether the deviation from the expected is significantly different, a

chi-square value was calculated (Table 2). With a 2 value calculated to be 2.3793 with

3 degrees of freedom, the probability that the deviation of the observed values from the

expected values was a chance occurrence is .5026. A probability greater than 0.05 is

not considered statistically significant; therefore, the deviation of the observed values

from the expected values must have occurred by chance. For a total of only 53 plants,

the 2 value calculated may not be reliable. The method in the characteristics of the

plants in the petri dish were interpreted by other groups are unknown. It may be that not

all plants were recorded, if say, they were not “alive” or not. This could be reason why

the observed data did not turn out as expected of the 9:3:3:1 ratio of a dihybrid cross.

While the 2 value calculated may not be reliable due to the low count of results,

these results do support that specific traits could be passed from one generation to the

next without phenotypically displaying that trait. This is important beyond the application

of crossing plants, since some traits that cause disorders can be carried by a parent and

passed on to the offspring without any knowledge of carrying that trait. The expansion of

knowledge in the field of genetics has greatly increased awareness of such genetic

disorders. And with the awareness of such genetic disorders, methods to decrease the

transmission of these disorders are also being investigated.

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References

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Brassica rapa (RBr) to linkage group R9. BMC Genet. 2007;8:64. Accessed at

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2048511/

Miko, I.. Gregor Mendel and the principles of inheritance. Nature Education

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mendel-and-the-principles-of-inheritance-593

Slankster, E., Chase J., Jones L., and Wendell D.. DNA-based genetic markers for rapid

cycling Brassica rapa (Fast Plants type) designed for the teaching laboratory.

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Vicedo M. The Chromosome Theory of Mendelian Inheritance: Explanation and Realism

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