Growth and Analysis of the Ceratopteris
(C-FERN) Life Cycle: Observing
Fertilization and Mature Gametophytes [1]
Jayanth (Jay) Krishnan
T.A. Ms. Bianca Pier
Lab Partner and Assistance Provided by
Ms. Catherine Mahoney
Section 1: Biology
November 29th, 2011
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Introduction/Purpose:
Why Did We Study This Problem?
I: The C-Fern Life Cycle
This multi-week genetics experiment involved sowing spores in nutrient-rich agar to
observe and analyze the life cycle of the autotrophic C-Fern. Furthermore, based on the analysis
done on the C-Fern life cycle, the lab class was able to make effective genetic predictions based
on inheritance patterns.
The life cycle of the fern starts with a single spore as most ferns are homosporous. The
spore is haploid meaning that it has half the amount of genetic material or half of the complete
set of chromosomes typically found in the organism. The spore develops into a heart-shaped
gametophyte (through mitosis) and survives using the process of photosynthesis. This phase is
the start of the gametophyte generation where all parts are haploid regarding genetic material.
Each of the gametophytes has both male and female sexual organs hence at this stage the
gametophyte has the properties of a hermaphrodite. The antheridium corresponds to the male sex
organ and similarly the archgonium corresponds to the female sex organ. These organs usually
mature separately allowing cross fertilization to occur between gametophytes. Fertilization
occurs from the sperm of the antheridium moving using their flagella to fertilize the egg of the
archgonium. When this happens a zygote, which is respectively diploid, is formed. [4]
Once the zygote has been created we experience an alteration of generations as the
fertilization in this cycle has been complete and the diploid will go through mitosis and meiosis
still without compromising its complete set of genetic material. The terminology of alteration of
generation also refers to switching from the multicellular diploid form, the sporophyte, the
multicellular haploid form, and the gametophyte.
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The fertilized egg now develops through mitosis into a sporophyte and the young plant
that grows out of the archgonium is a gametophyte. It is important to understand that the
sporophyte generation is always diploid while the gametophyte generation is haploid. With
further growth the fern grows and the underside of reproductive leaves contain clusters of
sporangia. The sporangia could then release spores through meiosis bringing us back to the 1st
step as we again move into the haploid generation. These spores would then again go through
mitosis to give rise to gametophytes. [4]
Figure 1: The Life Cycle of the C-Fern through a pictorial
representation. All the steps outlined above are visually depicted [3]
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II: Purpose of the Experiment:
In this experiment, we aim to observe the biology of sexual reproduction and the ideas of
Mendelian inheritance by observing and analyzing the life cycle of the C-Fern. To do this we are
observing the inheritance pattern of the mutant allele as you learn about the different stages of
the life cycle of the fern. We observed the many wild and mutant phenotypes for the
hermaphroditic gametophytes along with wild and mutant phenotypes for sporophytes.
I initially hypothesized that the mode of inheritance for the mutant allele or trait is
recessive. Based on this hypothesis, the expected proportion of the wild type to the mutant types
of hermaphrodite gametophytes is 1:1. The expected proportion of the wild type to the mutant
phenotypes of the sporophytes is 3:1.
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Materials and Methods
First and foremost, we needed to sow the spores on a 60 mm petri dish. The petri-dishes
all contained agar which is a layer of gel enriched with nutrients needed to promote C-Fern
growth. After labeling our petri-dishes, the area where the spores are to be sowed was wiped
with 70% ethanol. What we did next was take a sterile cotton tip applicator and touched the
spores at the bottom of a vial which we labeled as F1 spores. The applicator was then removed,
and the petri dish cover had been lifted. The spores were then flicked into a Petri dish,
containing agar, and the dish was covered with the lid. [2]
Between infrequently taking our observations, our prepared dish was kept in a culture
tray, among other dishes, covered with a dome top. Our TA, had placed this tray on a plant grow
stand with conditions that were corresponding to optimal growth. For example, the stand had a
full spectrum of light 24hrs/day; 7days/wk at a temperature between 28-30 degrees Celsius
Observations for this multi-week experiment were taken every Wednesday during our Biology
1010 Session 1 Lab Class (BIOL-1). [2]
During the first week of this experiment we simply sowed the spores, observed them
under a microscope, and took many pictures. When observed that many of the circular- haploid
spores had a visible striations alcross the trilete mark. A trilete mark is a three-fold (Y-shaped)
scar visible on the proximal side of some of the spore. Three lines radiate from the central pole.
During the next two weeks (12-14 days), our lab group observed clues that marked the
the initial development phases of the C-Fern. For example, at the two week mark, the mature
gametophyte was visible via a microscope. At this time, we took pictures of the wild and mutant
hermaphroditic gametophytes and the male gametophytes. During week three, after sowing had
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been done, we were able to observe fertilization. At this time, we took pictures of sexually
mature wild phenotypes and the mutant sporophytes.
The next step was to fertilize the gametophytes – a primary objective in this lab. In order
to do this we added distilled water to the plants so that the motile sperm, we observed under the
microscope, would be able to travel quite easily to the archegonia. If/when the fertilization is
successful a diploid sporophyte will begin to form and develop.
On week five, the last week of the experiment, the young sporophyte stage can be seen.
We then transfer these sporophytes to Premier Pro-Mix potting soil enriched with Osmocote
pellets. Four sporophytes we transferred had phenotypes that were mutant sporophyte on wild
gametophyte, wild sporophyte on wild gametophye, and mutant sporophyte on mutant
gametophyte. [2]
This concluded our final biology experiment.
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Results: Figures and Tables
Figure 2: Spore showing trilete mark. A trilete mark is a three-fold (Y-shaped)
scar visible on the proximal side of some of the spore. Three lines radiate from the
central pole.
Figure 3A: Mutant sporophyte on mutant gametophyte
Figure 3B: Wild sporophyte on wild gametophyte
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Figure 4A: Wild Male
Gametophyte
Figure 5: Mutant hermaphrodite
Figure 4B: Mutant Male
Figure 6: Wild hermaphrodite
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Figure 7: Wild sporophyte on mutant gametophyte
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Results: Tables
Table 1: Fertilization Events
Sperm Wild
Eggs Wild
Genotype of
P
sperm (P or p)
Genotype of egg
P
(P or p)
Genotype of
PP
diploid
sporophyte
Phenotype of
+
gametopyte (+ or
mutant)
Phenotype of
+
gametophyte (+
or mutant)
Appearance of
Wild/wild
sporo-/gamocombination
(e.g.,
wild/mutant)
Wild
Mutant
P
Mutant
Wild
p
mutant
mutant
p
p
P
P
Pp
Pp
pp
+
+
mutant
Mutant
+
Mutant
Wild/mutant
Wild/wild
Mutant/mutant
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Table 2: Phenotypes of Hermaphroditic Gametophytes
Wild
Phenotype
Mutant Phenotype
Observed
6
4
Expected
5
5
Difference
1
-1
0.2
0.2
(Difference)2
Expected
Chi square: 0.4
P-value: 0.527
Is this result significant? No
Should you accept or reject the null hypothesis (Ho)? Reject
Table 3: Phenotypes of Sporophytes – BIOL-1010 Data
Observed
Wild
Phenotype
Mutant Phenotype
2584
925
Expected
2631.75
877.25
Difference
-47.75
47.75
0.8664
0.8664
(Difference)2
Expected
Chi square: 1.73
P-value: 0.188
Is this result significant? No
Should you accept or reject the null hypothesis (Ho)? Accepted
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Table 4: Phenotypes of Gametophytes attached to Wild Sporophytes – BIOL1010 Data
Observed
Wild
Phenotype
Mutant Phenotype
1136
612
Expected
1165.33
582.67
Difference
-29.33
29.33
(Difference)2
0.738
0.738
Expected
Chi square: 1.476
P-value: 0.224
Is this result significant? No
Should you accept or reject the null hypothesis (Ho)? Accepted
Table 5: Phenotypes of Gametophytes attached to Mutant Sporophytes – BIOL-1010 Data
Observed
Wild
Phenotype
Mutant Phenotype
0
1124
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Results: Explanation
Out of the hermaphroditic gametophytes that we observed in this lab, six of them were
identified as wild gametophytes and four of them were observed as mutant gametophytes. Based
on these numbers, the proportion of observed hermaphrodite genotypes if 6:4 or 3:2. [Table 2]
What we expected to be the proportion of hermaphroditic gametophytes was noted as
1:1. Regarding the phenotypes of sporophytes we observed that there are a total of 2,584 wild
phenotypes and 925 mutant phenotypes. [Table 3]
Regarding the phenotypes of gametophytes attached to Wild Sporophytes, we observed
1,136 wild phenotypes and 612 mutant phenotypes. We expected the proportion to be 2:1.
Upon performing a chi-square test for comparison between these two tables of categorical
data we obtained a value was 1.476, with a P-value of 0.224 [Table 4]. Since we took our level
of significance to be .05 we concluded that this this p-value is insignificant and hence our null
hypothesis is presumed to be true.
Lastly regarding the phenotypes of Gametophytes attached to Mutant Sporophytes, we
observed 1,124 mutant phenotypes and zero wild phenotypes. We expected the proportion for
phenotypes of gametophytes attached to mutant sporophytes to be 0:1. What we observed indeed
correlated with what we expected and hence no statistical evidence for validity is required.
[Table 5] [1]
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Discussion:
From the results of our experiment we can properly conclude that the ferns that the spores
were sowed from were heterozygous as they produced a mutant allele. What the hypothesis
stated was that the mutant allele was recessive hence the expected proportion of the wild to
mutant gametophytes is 1:1.
However due to none of the chi-squared values we obtained were significant meaning
based on the p-values and therefore the null hypotheses should not be rejected. This states that
the observations and the expected values were not significantly different. Our initial
prediction in the introduction was confirmed that the mutant allele is proven to be recessive.
When we hypothesized about the inheritance pattern, the expected proportion of wild to mutant
sporophytes is 3:1. In concordance with that hypothesis, the expected proportion of the
sporophyte-gametophyte combination was 2:1. None of the P-values were significant meaning
that the observed results did indeed bolster our original presumption or the initial hypothesis. [1]
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Citations
[1] Radlick,L. Grading Rubric: Growing C-Ferns. BIOL 1010. Spring 2001
[2] Radlick, “Introducing C-fern (Ceretopteris richardii). “Observing Fertilization and Mature
Gametophytes. 2011. Print.
[3] "C-Fern Life Cycle." Blog Spot. Web
[4] Campbell, Neil A. “Plants, Fungi, and the Colonization of Land.” Biology Concepts and
Connectoins. 6th ed. San Francisco, Calif. [u.a.: Pearson Education, 2009. 347. Print.
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Attachments:
1) Student Workbook
2) Fern Cycle Pictorial Representation
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