A Study of C-fern Genetics
By: Jennifer Harper
and
Valerie Friedmann
C-Ferns
• C-ferns are a hybrid of
species found in Cuba
and Nicaragua. The
strain of DNA that causes
the ferns to have a short
life cycle was taken from
one fern and introduced
into the genes of the other
fern by means of
repeated cross breeding.
• The abbreviated name Cfern comes from
Ceratopteris richardii.
• Found in tropical areas of
the world.
• Semi-Aquatic
C-ferns
• Exhibit both haploid and diploid phases.
Problem
• The researchers wished to learn more about
genetics and the way traits are passed from one
generation to the next.
• F1 generation polka dot C-ferns
will be cross bred to create an
F2 generation of plants.
Wild Type Hermaphrodite
C-fern
C-Ferns
• The growth from
spore to gametophyte
takes only 2 weeks.
• From fertilization, it
takes another 2
weeks to produce a
sporophyte.
• In 90 days, the fern is
fully mature.
What is a polka dot C-fern?
• The polka dot characteristic is a mutation
that was induced using an EMS mutagen.
• The chloroplasts clump together in the
fern cells to create the look of polka dots.
• When mature, polka dot C-ferns will
appear more pale than wild type C-ferns,
but will still grow and reproduce the
same.
Wild Type
Polka Dot
Hypothesis
It is expected that, because
the polka dot trait is believed
to be recessive, the crossing of
two mutant forms of polka
dot C-fern plants will produce
only polka dot offspring.
Procedure
• The first attempt at
producing Polka dot Cferns was made by using
spores that had been
subjected to an EMS
mutagen.
• EMS causes many types
of mutations in C-ferns,
not only polka dots.
• Some other mutations
are pale, lethal, dwarf,
ruffled, and bubbles.
• The plates were sown
and the gametophytes
were searched through
only to find that not
enough polka dot Cferns had been produced
to continue the
experiment and acquire
accurate results.
Because the ferns sewn
at UT could not be used
in the project, they were
used to provide the
researchers with basic
knowledge of C-fern
maintenance.
• Temperature control
28ºC
• Medium Production
solidification
• Sterilization
contamination prevention
• After the first unsuccessful attempt to
produce polka dot C-ferns, spores were
purchased from Carolina Biological
Supply.
• These spores were an F1 hybrid of
polka dot and wild type C-ferns in a
1:1 ratio.
• Growth of the C-fern
spores was
accelerated using the
“Dark Start”
method.
• The spore vial was
inoculated with 5 mL
of distilled water and
covered completely
with aluminum foil.
• Because
hermaphrodites
mature more rapidly
than males, 32 plates
were sown and
labeled “male” and
two days later 32
more plates were
sown and labeled
“female.”
• Once gender was
• All wild type ferns
visible under the
were removed from
stereoscope, male
both male and female
ferns were removed plates.
from the
• This was done to
hermaphrodite
prevent unwanted
plates.
fertilization from
• Hermaphrodites
occurring.
were removed from • All ferns were counted
the male plates.
and recorded for
comparison.
• C-ferns are a
homosporous plant,
meaning that a single
spore can become male
or hermaphrodite
depending on climate
and the pheromone,
antheridiogen.
• Because Antheridiogen
is secreted by
developing
gametophytes, it affects
only the gametophytes
that develop later.
• C-ferns grown in the
presence of the Ace
pheromone develop
antheridia, which are
the male reproductive
organs.
• Once the ferns were
about two weeks old
and had reached
sexual maturity,
sperm release buffer
(SRB) was added to
the male plates to
induce the release of
sperm in male Cferns.
• Water can also be
used to release
sperm.
• This sperm was
taken from the males
using a sterile pipette
and placed at the
archegonia of the
hermaphrodite ferns.
Archegonia, the female reproductive organs,
contain one egg each that lies at the base of a
small neck. The neck, which protrudes out from
the surface of the gametophyte, consists of four
rows of cells and neck canal cells in the middle.
When archegonia are mature, water may be
added to the ferns to cause the neck canal cells
to burst open forming a small open canal
leading to the egg. The contents of the neck
canal cells are deposited at the neck as an
archegonial discharge that attracts the sperm to
the top of the open neck.
Egg
Neck Canal
Archegonia
• Once fertilized, the archegonia swell and by
mitotic cell division, a diploid embryo is
formed.
• After one to two weeks, the embryo has
developed into a sporophyte, identified by the
rhizome, a short upright stem with roots and
leaves.
• Sporophytes produce an unlimited number of
genotypically identical spores.
Results
F1 Gametophyte Phenotype
760
750
740
730
720
710
700
690
680
670
660
753
729
Polka Dot
Wild Type
Results
F2 Sporophyte Phenotype
220
210
200
190
180
170
160
150
140
130
120
110
100
216
111
Polka Dot
Wild Type
Conclusion
• Because the F2
generation was a 1:2
ratio of polka dot to
wild type C-ferns, it
was believed that
human error
occurred and that
wild type sperm was
present during
fertilization.
• With further
research, it was
discovered that the
polka dot trait could
be present on two
different genes.
• This would mean
that the Polka dot
trait is not a simple
trait.
Research Improvements
• The hermaphrodites
would be removed
from their original
plates and isolated in a
new plate as soon as
gender and phenotype
is evident.
• This would prevent
any wild type sperm
from being present
during fertilization.
• Temperature would be
regulated in the
growth pods before
ferns were placed in
them to ensure that
maximum growth
potential can be
achieved.
• Researchers must be
extremely patient in
order to find the polka
dot ferns amongst the
wild type ferns.
Further Studies
• Further studies are being
done to increase
knowledge of the
location of the polka dot
trait.
• Two types of polka dot
ferns will be cross bred.
• One type of fern has
more condensed
chloroplasts than the
other.
• If in crossing these two
kinds of polka dot ferns
we receive all polka dot
ferns, we can conclude
that the trait is on the
same gene.
• If we receive wild type
ferns, assuming that
human error has not
occurred, we can conclude
that the trait is on at least
two different genes.
Acknowledgements
• We would like to thank the Tennessee Junior
Academy of Science for generously providing
us with the finances needed to perform this
experiment.
• We would also like to express our gratitude to
Dr. Les Hickock and Stephenie Baxter for
mentoring us and for making the UT facilities
available to us, as well as allowing us to use the
pictures on his website.
• Additionally, we would like to thank Mrs. Jan
Coley for motivating us, providing us with
constant guidance, and teaching us to dig
deeper into the world of science.