C-Fern® Web Manual
LESLIE G. HICKOK AND THOMAS R. WARNE
CURRENT CONTACT INFORMATION
For more information about using C-Fern –
http:// www.c-fern.org
Leslie G. Hickok - cfern@utk.edu
Thomas R. Warne – warne@c-fern.org
COPYRIGHT AND REVISIONS
2009, Revised and updated
©2008-present, Leslie G. Hickok and Thomas R. Warne
2004, Revised and updated
©1998-2008, University of Tennessee Research Foundation
ACKNOWLEDGMENTS
Development of C-Fern was supported, in part, by the
National Science Foundation and the University of
Tennessee. We gratefully acknowledge Stephenie Baxter for
her superb laboratory and greenhouse assistance as well as
illustrating the developmental stages of C-Fern. We also
thank Jennifer Panter and Dr. Dale L. Vogelien for their
contributions towards the optimization of Ceratopteris
culture.
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Table of Contents
Introduction...................................................................................5
Why Use C-Fern®?........................................................................................................ 5
C-Fern and the National Science Standards.................................................................6
What Is Ceratopteris? ...................................................................................................8
Taxonomy...................................................................................................................... 9
The C-Fern Life Cycle ................................................................................................ 10
A Perspective on Plant Reproduction— C-Fern and Teaching the Concept of
Alternation of Generations ..........................................................................................14
Culture Instructions for C-Fern® Investigations .....................18
Introduction.................................................................................................................. 18
Quick Start .................................................................................................................. 18
Culture Instructions .....................................................................................................19
1. Getting Started.................................................................................................... 19
2. Determining Sowing Density and Number of Cultures....................................... 20
3. Preparing the Culture Medium............................................................................ 20
A. Using Pre-Made Bottled C-Fern Medium ...................................................... 20
B. Using Powdered Basic C-Fern Medium ....................................................... 21
C. Using a Microwave to Prepare C-Fern Powdered Medium...........................24
D. Preparation of Basic C-Fern Medium From Stock Solutions......................... 25
4. Inoculating (Sowing) Cultures Using Presterilized C-Fern Spores..................... 27
Seven Good Habits for Sowing C-Fern Cultures ....................................................... 27
5. Maintaining C-Fern Cultures............................................................................... 29
A. Culture Domes................................................................................................29
B. Growth Pod.....................................................................................................29
C. Temperature................................................................................................... 31
D. Light ............................................................................................................... 31
i. Growth Pod Requirements.......................................................................... 31
ii. Culture Dome Requirements ..................................................................... 31
iii. Constructing a C-Fern Light Stand ........................................................... 32
6. Observing Cultures..............................................................................................33
7. Sporophyte Culture............................................................................................. 34
A. Standard C-Fern (RN cultivar) Sporophytes.................................................. 34
B. C-Fern Express Sporophytes......................................................................... 35
8. Additional Information For Educators..................................................................36
C-Fern® Web Manual
3
A. Hints for Large Classes.................................................................................. 36
B. Independent Student Research Projects....................................................... 37
C. Questions for Discussion................................................................................38
Additional Culture Methods and Techniques..........................41
Surface Sterilization of C-Fern Spores .......................................................................41
DarkStart Method ..................................................................................................... 41
Dry Sowing of Presterilized C-Fern Spores................................................................ 44
Liquid Culture of C-Fern Gametophytes...................................................................... 44
Mutagenesis of C-Fern Spores................................................................................... 44
1. Ethyl Methane Sulfonate (EMS) Mutagenesis.................................................... 45
2. X-Ray Mutagenesis............................................................................................. 46
Controlled Fertilization: Selfing and Crossing Techniques ........................................ 47
1. Self-Fertilization...................................................................................................47
2. Cross-Fertilization............................................................................................... 48
Preparation of Semipermanent Slides for Observation and Analysis of C-Fern
Gametophytes ............................................................................................................ 49
Greenhouse Culture of C-Fern Sporophytes.............................................................. 51
1. Transplantation....................................................................................................51
2. Fertilization.......................................................................................................... 52
Cloning of C-Fern Sporophytes ..................................................................................52
Collection of C-Fern Spores........................................................................................ 53
Sport Reports - Descriptions of C-Fern Mutants....................55
Glossary of Selected Terms...................................................... 63
Bibliography................................................................................68
User Service................................................................................77
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Introduction
Introduction
A
re plants alive? Do they do anything really interesting? Ask
these questions to any number of high school or college
students and be prepared for a variety of answers! Even
biology majors may give surprising responses! It is no secret that
teaching the basics of plant biology and capturing students’ interest
can be a daunting task. To most students, plants are just not as
naturally interesting or visually compelling as animals are.
Nonetheless, plants are integral and essential components of our
living world, and learning about them is a necessary part of any
liberal or specialized education experience. Finding more effective
ways to teach about them is a challenge.
Why Use C-Fern®?
Simple and Complex Development
• haploid (gametophyte) and diploid (sporophyte) phases
• cellular and whole plant observations and experiments
Rapid Development
• spore to sexually mature gametophytes in 14 days
• spore to spore in less than 90 days (original C-Fern) or 60 days (C-Fern Express)
Direct Observation of Development Haploid/Diploid
Genetic System Optimized for Classroom Use
• inexpensive
• safe
• miniaturized
• very large numbers of individuals and treatments possible
The advantages of using C-Fern® as a model plant system for
teaching derive from the unique properties of its development and
life cycle. Both haploid (gametophyte) and diploid (sporophyte)
C-Fern® Web Manual
5
Introduction
phases exist independently; therefore, observations and
experimental studies are possible at both the cellular and whole
plant levels without artificial manipulations of the life cycle.
Development from spores to sexually mature gametophytes to
young sporophytes can be observed within 2 to 3 weeks. The
complete, spore-to-spore life cycle can occur in less than 90 days.
The rapid growth and small size of gametophytes allows for the
miniaturization of experiments, such that large numbers of
individuals and treatments can be used in a small space and short
time. The haploid/diploid genetic system is very simple. C-Fern
combines the features of both higher and lower plant systems. The
developmental simplicity and haploid nature of the independent
gametophytic phase provides opportunities that are not available in
angiosperm models. At the same time, the ability to study many
processes within the complex vascular sporophyte phase allows
direct comparisons with higher plant systems (e.g., Wisconsin Fast
Plants™, Arabidopsis) that are not possible with developmentally
simpler systems such as mosses (e.g., Funaria, Physcomitrella) or
algae (e.g., Chlamydomonas, Euglena).
This manual presents:
• The protocols necessary to successfully culture and use C-Fern
in educational settings.
• Background information about the biology and natural history of
Ceratopteris (the fern from which the C-Fern materials derive).
C-Fern and the National Science Standards
Laboratory-based education can serve many functions, such as the
reinforcement and illustration of lecture and text material and the
exposure of students to the uses of sophisticated equipment and
current methodologies. However, an equally important and often
neglected function is the use of the biological laboratory component
to promote general literacy in the scientific method; i.e., how the
process of science works, what constitutes scientific information,
and how scientific information is acquired—in short, how to do
science. Comprehension of the scientific method involves
understanding and applying a large set of interrelated skills—
problem identification, hypothesis formulation, experimental design,
implementation, data collection, analysis and synthesis,
communication of results—that necessitate different approaches to
teaching. The National Science Education Standards (National
C-Fern® Web Manual
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Introduction
Table 1. Alignment of C-Fern with the National Research Council Life Science Standards. C-Fern is
readily applicable to fundamental concepts or principles that underlie each of the following topics. The
bulleted list (•) below each topic notes some specific C-Fern attributes that are relevant to each category.
National Research Council Life Science Standards
Levels K–4
Characteristics of organisms
plant form and function
basic requirements for plant growth
Life cycles of organisms
sexual and asexual reproduction
non-flowering plant reproduction
alternation of generations
Organisms and environment
Levels 5–8
Levels 9–12
Structure and function in living
The cell
systems
structure and function in gametophyte
C-Fern exists as two independent
organisms
gametophytes allow easy visualization
of cell structure and differentiation
comparative studies of gametophyte
and sporophyte structure and
function
Reproduction and heredity
tissues—vegetative, meristematic,
absorptive / anchoring, specialized
sperm cells
cell motility (sperm)
chemotaxis (sperm)
spores as single-celled propagules
Molecular basis of heredity
C-Fern clearly shows the relationship although structure and function are
of all aspects of sexual reproduction very different, C-Fern gametophytes
and sporophytes contain the same
genetic studies with C-Fern allow
genetic instructions
examination of both haploid
(gametophytic) and diploid
gametophytes have half the
(sporophytic) ratios
chromosomes of the sporophyte
a wide variety of mutant stocks allows sexual differentiation in gametophytes
extended investigation of the effects is related to a chemical control of
of mutation
gene expression
mutants exist that have altered
responses to the chemical
(pheromone) that controls sexual
type
Regulation and behavior
responses to changes in environment changes in sex ratio in response to
(C-Fern can be grown both
population size and/or environment
terrestrially and aquatically)
changes in sporophyte growthin
response to aquatic vs. terrestrial
environment
Populations and ecosystems
the effect of population size on sex
ratio or early sporophyte
development
Diversity and adaptation of
organisms
the structure and function of the CFern life cycle relative to other
plants
adaptation to different environments
Biological evolution
ferns have adapted for a specific lifestyle
C-Fern has specialize dadaptations
for a “semi-aquatic” life-style
Matter, energy, and
organization of living systems
C-Fern is autotrophic and exhibits
distinct adaptations for
photosynthesis and related activities
C-Fern gametophyte and sporophyte
generations are independently
autotrophic
Behavior of organisms
chemotaxis by C-Fern sperm shows
behavior at the single cell level
C-Fern shows typical plant responses
to the environment
population pressures influence
development in both gametophytes
and sporophytes
C-Fern® Web Manual
7
Introduction
Committee on Science Education Standards and Assessment; and
National Research Council 1996) clearly reinforce the need to more
effectively teach the process of science through inquiry. However,
normal time constraints and equipment needs can make it difficult
to implement such an approach. One option is to introduce
exercises in experimental science using an amenable and flexible
model organism relevant to a broad range of disciplines and subject
areas. In plants, only a few model systems have attributes that
make them sufficient to meet the demands of a wide range of
situations and levels. C-Fern is a broadly useful model system that
possesses a suite of characteristics and developmental features
that make it ideal for use in both the classroom and in student
initiated research. The broad applicability of C-Fern for a variety of
classroom uses means that it can be applied and integrated into
several of the Content Standards. Life Science standards focus on
the science facts, concepts, principles, theories, and models that
are important for students to know, understand, and use.
What Is Ceratopteris?
Ceratopteris is a genus of homosporous ferns found in most
tropical and subtropical areas of the world (Lloyd 1973). Species
grow as either aquatics or subaquatics and are restricted in habitat
to ponds, rivers, or intermittent wet areas such as ditches, rice
paddies, or taro patches. Although some require an aquatic habitat,
most species can be successfully grown in standard greenhouse
pot culture under warm, humid conditions. Ceratopteris is eaten in
areas of Southeastern Asia, and there was once an attempt to
develop it as a crop in the Philippines (Copeland 1942). Currently,
commercial applications are primarily limited to its use as an
aquarium plant, where it is sold under the common name of water
sprite and has even been immortalized in plastic replicas. In some
environments, Ceratopteris can become an aggressive weed by
clogging up freshwater streams and drainage systems.
Instead of the long-lived rhizome and perennial habit of most
homosporous ferns, Ceratopteris has a short, upright rhizome and
grows as an annual. The fronds form small “fiddleheads” when
young and are dimorphic at maturity. Initial fronds are sterile and
from simple to three-lobed, but as development proceeds the
fronds become increasingly more dissected and fertile. Sporangia
occur in continuous rows on veins along the ventral edges of fertile
fronds and are covered by the inrolled margin of the frond, forming
C-Fern® Web Manual
8
Introduction
a “false indusium.” In addition to sexual reproduction via meiotic
production of spores and subsequent gametophytes, Ceratopteris
sporophytes have a prolific capacity for vegetative reproduction.
Buds found in the axes of subdivisions of the frond can develop
rapidly into plantlets and are likely the reason for its weedy nature
in some habitats. Both in nature and under greenhouse culture, it is
not unusual to find senescent fronds abundantly covered with
developing plantlets that originated from these buds. On a practical
level, the buds provide a convenient means for vegetative
propagation of particular genotypes.
Taxonomy
Homosporous ferns like Ceratopteris belong to the order Filicales,
within the class Filicopsida, in the division Tracheophyta, kingdom
Plantae. Because Ceratopteris shows a variety of morphological
characters that are exhibited by a number of different families,
phylogenetic affinities at the family level are unclear. Some
taxonomic treatments have placed Ceratopteris in its own
monotypic family, Parkeriaceae, while others have included it as a
subfamily or tribe within the Pteridaceae (Tryon and Tryon 1990).
Robert M. Lloyd (Lloyd 1974, Lloyd 1993), in his taxonomic
treatments, recognized four species: C. thalictroides, C. richardii,
C. pteridoides, and C. cornuta. Ceratopteris thalictroides is
described as a highly polymorphic species with pantropical
distribution. Ceratopteris pteridoides and C. cornuta, show some
similarities to each other but are both morphologically distinct from
C. thalictroides. Ceratopteris pteridoides, typically thick-leafed and
floating, is principally limited to Central and South America, while
C. cornuta is mainly confined in distribution to mainland Africa. The
fourth species, C. richardii, is morphologically similar to C.
thalictroides; the only consistent difference noted by Lloyd (Lloyd
1974, Lloyd 1993) was the 16-spored sporangia of C. richardii as
opposed to the 32-spored sporangia of all other species. Because
of the plants’ similarities, Tryon and Tryon (1982) did not recognize
C. richardii as being distinct from C. thalictroides. However,
cytological observations show that C. thalictroides is tetraploid with
n=77,78 and the other three species recognized by Lloyd are
diploid with n=39 (Hickok 1977, Hickok 1979). Analyses of
synthesized interspecific hybrids indicate incomplete reproductive
isolation among the diploid species. The presence of herbarium
specimens with both 16- and 32spored sporangia and with mixed
C-Fern® Web Manual
9
Introduction
features of both C. richardii and C. pteridoides suggest that
hybridization occurs naturally (Lloyd 1993).
Although a number of homozygous diploid and tetraploid
accessions of Ceratopteris are available, the Hn-n strain has been
used in most experimental studies (Hickok 1987, Hickok et al.
1995, Hickok et al. 1987). Hn-n is a diploid (n=39) derived from a
collection of C. richardii from Cuba (Killip 44595 GH). In addition,
two other diploid strains, 176D and ΦN8, are also in current use
(see Hickok et al. 1995). All three strains show sexually dimorphic
gametophytes, i.e., males and hermaphrodites, with males
developing in response to the pheromone antheridiogen (ACe).
However, 176D shows only slight sensitivity to antheridiogen as
compared to Hn-n and ΦN8, and it consequently has fewer males
in multispore cultures. The three strains can be crossed readily.
Hybrids between Hn-n and 176D show ca. 90% spore viability
while crosses of ΦN8 with either Hn-n or 176D show ca. 30%
spore viability. During the past several years, our laboratory has
developed an improved diploid cultivar, RN, from a series of
crosses and backcrosses. All of the exercises presented here are
based on the use of the RN cultivar.
The C-Fern Life Cycle
Like all homosporous ferns, C-Fern has two independent
autotrophic phases: a structurally simple, haploid gametophyte and
a vascular, diploid sporophyte. The gametophytic phase, which
develops mitotically after germination of the single-celled spore,
can be cultured axenically on a simple inorganic medium.
Development of this haploid phase is very rapid. Germination
occurs 3–4 days following inoculation, and full sexual maturity is
attained within 6–8 days from germination. At maturity, the
gametophyte consists of a small (2 mm), simple, essentially twodimensional thallus with rhizoids, vegetative cells, and sexual
organs (archegonia and antheridia). The archegonium is the female
organ; it contains one egg that lies at the base of a small neck
sticking out from the surface of the gametophyte. The neck consists
of four rows of cells, along with a few “neck canal cells” in the
middle. The antheridium is the male sex organ; each contains 16
sperm. In the presence of water, the neck canal cells in mature
archegonia burst open, creating a small, open canal leading to the
egg. The canal cells’ contents are deposited near the top of the
C-Fern® Web Manual
10
Introduction
open neck. Meanwhile, the antheridia are also active. In the
presence of water they also burst open, discharging motile sperm
(spermatozoids). The rapidly swimming sperm are irresistibly
attracted by the discharge from the archegonium. In a few minutes,
hundreds of sperm can be seen swarming around the neck of the
archegonium, and one of them eventually wiggles its way down the
neck and fertilizes the egg. After fertilization of the egg, the
resulting diploid zygote develops rapidly by mitotic cell division,
forming an embryo. Embryos are clearly visible after a few days,
and in only 1–2 weeks roots and leaves can be seen on the small
diploid sporophytes. The gametophyte soon dies and the
sporophyte grows to maturity. It undergoes meiosis and produces
spores to continue the life cycle.
The pheromone-like substance, antheridiogen (ACe), secreted by
developing gametophytes controls differentiation of two distinct
sexual forms of gametophytes. ACe is likely biosynthetically related
to gibberellins and is effective at extremely low concentrations
(Warne and Hickok 1989). In the absence of ACe, gametophytes
develop initially as heart-shaped (cordate) females with archegonia
and subsequently as hermaphrodites with both archegonia and a
few antheridia. In hermaphrodites, a defined meristematic region
(notch meristem) is present, and growth is indeterminate until
fertilization of an egg occurs. Meristematic activity ceases shortly
after fertilization. In contrast to hermaphrodites, gametophytes that
mature in the presence of ACe develop into tongue-shaped males
that are small, determinate, lack a meristem, and produce large
numbers of antheridia.
At the vascular sporophyte stage, a C-Fern consists of a short
upright stem (rhizome) with roots and leaves (fronds) and reaches
a height of 10–40+ cm. In contrast to many ferns, the Ceratopteris
sporophyte is not woody and grows rapidly as an annual. Spore
production via meiosis occurs within sporangia that are located on
the margins of fertile leaves. Upon maturity, spores are produced
continually and are unlimited in number. Compared to many ferns,
spores are quite large (ca.120 µm) and relatively easy to handle.
Because individual haploid gametophytes can be self-fertilized,
sporophytes completely homozygous across all genetic loci can be
produced in one generation of selfing. Such sporophytes produce
an unlimited number of genetically identical spores. If kept dry,
spores remain viable for many years.
C-Fern® Web Manual
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Introduction
B
D
A
C
Figure 1. C-Fern spores. A,B — ungerminated, proximal (A) and distal (B) views; C,D — germinated,
showing splitting along trilete mark (C) and emergence of primary rhizoid (D). Spore diameter ca. 120 µm.
Notch meristem
Archegonium
Figure 2. Young C-Fern
gametophyte, 5 days from start
(DFS) of culture. Spore coat diameter
ca. 120 µm.
Antheridium
Egg
Sperm
Figure 3. Mature hermaphroditic C-Fern gametophyte with archegonia behind
the notch meristem and a single antheridium on the margin, ca. 10 DFS. Spore
coat diameter ca. 120 µm. Close-up: view of mature archegonium during
fertilization. Sperm enter the open neck canal, uncoil, and move toward the egg.
C-Fern® Web Manual
12
Introduction
Mulitlayered
structure
Flagellum
Dehisced
antheridium
Sperm body
Figure 5. Individual mature
sperm, ca. 8.8 × 5.5 µm.
Undehisced
antheridium
Sperm
De-differentiated
meristem region
Figure 4. Mature male C-Fern
gametophyte, ca. 10 DFS. Sperm are
released from the numerous antheridia on
the surface of the male gametophyte.
Spore coat diameter, ca. 120 µm.
Young
embryo
Immature
archegonium
Young
embryo
Mature
archegonium
Region of old
archegonial neck
Figure 7. Close-up of hermaphroditic C-Fern
gametophyte, ca. 3 days after fertilization, showing
unfertilized (immature and mature) archegonia and
young embryo developing within proliferated
archegonial tissue. The remains of the old archegonial
neck can be seen at the distal end of the embryo.
Figure 6. Hermaphroditic C-Fern
gametophyte, ca. 3 days after
fertilization, showing a young
sporophyte embryo. The embryo
is covered by proliferated
archegonial tissue. Following
fertilization, cell division ceases in
the notch meristem region of the
gametophyte and cells enlarge.
As the sporophyte continues to
develop, the gametophyte
eventually dies. Spore coat
diameter, ca. 120 µm.
C-Fern® Web Manual
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Introduction
A Perspective on Plant Reproduction— C-Fern and
Teaching the Concept of Alternation of Generations
Like other organisms, plants have to reproduce in order to survive
as a species, increase in number, and colonize new habitats.
Plants reproduce by two basic methods: asexual and sexual
reproduction. Generally, the most efficient form of reproduction is
asexual. This form typically does not involve a partner and allows
an individual plant to produce duplicate copies of itself. Common
examples of asexual reproduction can be seen in the ability of new
plants to arise from stem cuttings, the production of “baby” plants
from specialized buds on the mother plant (e.g., C-Fern and
Kalanchoe, or “Mexican hat plant”) and specialized outgrowths from
the mother plant, such as potato tubers or strawberry runners. In
asexual reproduction, the offspring are genetically identical to the
mother plant.
Although asexual reproduction is an important aspect of plant
biology, sexual reproduction is considered an essential activity of
most types of plants. This is because during sexual reproduction
the genes are shuffled to produce new combinations in the next
generation. This genetic variation has been important in the
successful evolution of plants over millions of years. It is also
important to human agriculture because it allows the combination
and modification of specific traits during the breeding of crop plants.
Also, an important portion of our diets consists of the direct
products of sexual reproduction—fruits and seeds.
Sexual reproduction can involve either one or two plants. When a
single plant is involved, different organs develop on the same plant
to produce male and female gametes. When two plants are
involved, one produces male and the other female gametes. Just
as in animals, these gametes can be referred to as egg and sperm.
The fertilization of an egg by a sperm to produce a zygote is a
critical event in the reproductive process.
An important difference between most plants and animals is that
gametes are not the immediate products of meiotic cell division, as
they typically are in animals. Instead, meiosis in plants yields
spores. Spores develop into gametophytes that in turn produce
gametes. Union of the gametes (fertilization or syngamy) results in
a diploid embryo that gives rise to a mature sporophyte. Mature
sporophytes produce spores via meiotic division. This cycle of
C-Fern® Web Manual
14
Introduction
gametophyte to sporophyte, each with its own role in the process of
sexual reproduction, is commonly referred to as the alternation of
generations. This reproductive complexity, while advantageous for
the plants, can be a difficult concept for students to understand.
Nevertheless, there’s a good reason for the added step! Because
plants are for the most part stationary, they have developed a
different approach than animals to the problem of getting opposite
sexual types together to mate. The solution? Spores! As opposed
to the direct meiotic production of vulnerable gametes, spores have
an increased potential for transport and for survival over extended
periods of time and in a variety of environmental conditions. They
are suited to a plant’s mode of existence, where a mate may not be
immediately available or close at hand when meiosis occurs. When
conditions are favorable, meiotically-produced spores give rise to
gametophytes and, subsequently, to gametes. To complete the
process, plants have evolved a variety of fascinating ways to
ensure that fertilization occurs!
In C-Fern, students can directly observe the sexual reproductive
process and the alternation of generations. The development of
gametophytes and sporophytes, production of sperm and eggs, and
fertilization can all be easily seen. Although ferns are typically
known only by the conspicuous, green, leafy, sporophyte stage, this
stage represents only a portion of the sexual life cycle. Meiosis
occurs in ferns’ sporangia, borne on the sporophyte, sometimes on
the underside of the sporophyte leaf. In contrast to the microspores
and megaspores of flowering plants and heterosporous ferns,
homosporous ferns such as C-Fern produce only one type of spore.
When they are ripe, spores are released from the sporophyte, and,
if they land in a suitable environment, they germinate and develop
into small (1–2 mm) green, free-living gametophytes. Fern
gametophytes may develop as hermaphrodites with both antheridia
and archegonia or as distinct male plants with antheridia. When
mature and in the presence of water, antheridia burst open and
release the sperm cells, which swim in search of a receptive egg. In
the archegonium, the egg is located at the end of a short, closed
tube or neck. When mature and when water is present, the neck
opens and spews out cellular material, including a chemical
attractant that draws masses of sperm to the neck opening. The
sperm then attempt to swim down the neck and fertilize the egg.
After fertilization by a single sperm, the zygote develops by mitotic
divisions into a young embryo that can be seen within the now
“pregnant” archegonium. Continued development produces a leafy
C-Fern® Web Manual
15
Introduction
green sporophyte to complete the sexual cycle. All of these stages
are clearly observable in C-Fern cultures and are explored fully in
the C-Fern investigations dealing with reproduction and
differentiation. An understanding of alternation of generations
gained from study of the C-Fern life cycle can help students
understand the life cycle of flowering plants.
In flowering plants, the alternation of generations is more difficult for
students to observe. This is because the male and female
gametophyte generations have been reduced through evolution to
only a few cells. Meiotically produced microspores mature into the
pollen, contained within the stamen. The pollen grain contains the
much-reduced male gametophyte, consisting of only two cells at
maturity. When pollen is transferred to a compatible stigma, the
pollen tube grows down the style of the flower. When it reaches the
vicinity of the egg, a mitotic division produces sperm. In the
developing ovary, meiosis results in a megaspore that then
develops by mitotic division into a much-reduced female
gametophyte, the embryo sac. The embryo sac contains the egg,
along with other nuclei important to the process of embryo
formation and seed development. The production of pollen, which
can be easily transported, allows mating (pollination) by a variety of
mechanisms, including wind, water, insects, and other animals. The
specialization of many types of flowering plants, with their intricate
adaptations to ensure sexual reproduction, has resulted in a
diverse and fascinating group of organisms. If students’ concepts of
plant sexual reproduction are first thoroughly grounded in the
dynamic representation of alternation of generations represented
by C-Fern, they will better understand the flowering plant life cycle
as well.
C-Fern® Web Manual
16
Introduction
Figure 8. Life Cycle of C-Fern
C-Fern® Web Manual
17
Culture Instructions for C-Fern® Investigations
Culture Instructions for C-Fern ® Investigations
Introduction
C
-Fern®is an exciting and unique organism that is easy to
grow using inexpensive materials and simple growing
conditions. To help you get started quickly and easily, follow
the Quick Start instructions below. To ensure your success with CFern, additional detailed information and options are included in the
subsequent sections of this document. Please also see the C-Fern
Web site (http://www.C-Fern.org).
All of the INSTRUCTIONS presented here are based on the use of
the original C-Fern (RN) cultivar. A new cultivar, C-Fern Express,
was released in 2009. C-Fern Express was derived from crosses
between two Japanese strains of C. thalictroides and exhibits a
shorter life cycle time (<60 days) and smaller sporophyte size. With
the exception of sporophyte culture, all methods developed for RN
are applicable to C-Fern Express.
C-Fern branded materials, supplies, teaching aids and
investigations are available from distributors of C-Fern products.
Quick Start
C-Fern® and C-Fern Express cultures are started from spores.
Surface-sterilized spores of the wildtype and mutant stocks are
available through distributors in 10-mg units. For general use, 4 mL
of sterile water should be added to a vial to make a suspension of
spores. Spores are then inoculated into 60- × 15-mm petri dishes
containing Basic C-Fern Medium. Medium is available pre-made or
in powder form. Instructions for preparing the medium are detailed
in this manual. Use a sterile, disposable transfer pipet to inoculate
each petri dish with 3 drops of the spore suspension. This will
generate up to 36 cultures with a standard density of >300 spores
per dish. Spores should be spread evenly over the agar surface
using a sterile bent paper clip or microbial spreader. It is very
important to maintain cultures under continuous light and at a
temperature of 28°C (82°F). These conditions are easily achieved
by using a 15-W screw-in fluorescent bulb to illuminate cultures
within a C-Fern Growth Pod™. This setup is described in these
instructions and on the Web (http://www.C-Fern.org). Under these
C-Fern® Web Manual
18
Culture Instructions for C-Fern® Investigations
conditions, development will proceed as depicted in Figure 8. If the
culture temperature is cooler and/or constant illumination is not
provided, development will be slower.
For C-Fern Express cultures, follow the same basic culture setup
as described for original or standard C-Fern. Please take note of
the following differences of C-Fern Express. If cultured at 82 ºF,
mature gametophytes can be observed at 10 days from sowing
spores. While standard C-Fern gametophytes have clear
distinctions between the small males and larger hermaphrodites, CFern Express gametophytes are more variable. The basic
distinction between the smaller males (no meristem and containing
many antheridia) and larger hermaphrodites (a meristem,
archegonia and few antheridia) remains. However, some smaller
C-Fern Express gametophytes with several antheridia may initially
look like males, but subsequently develop a meristem and become
hermaphrodites with several antheridia.
Water added to gametophyte cultures at 10 days or later will allow
fertilization by swimming sperm. One week later, young
sporophytes will be visible at the first leaf stage.
Culture Instructions
1. Getting Started
Detailed written exercises are available from C-Fern suppliers and
contain both Teacher and Student versions.
Dried C-Fern spores are long lived and are available from C-Fern
suppliers in small plastic vials, either as pre-sterilized 5- or 10-mg
units or as unsterilized 40-mg units. The pre-sterilized spores are
very convenient and easy to use— just add the specified amount of
sterile water and sow! If using unsterilized spores, the sterilization
procedure is simple and efficient (refer to Step-by-Step Procedure
for Spore Sterilization in the Surface Sterilization of C-Fern Spores
Section on page 41).
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Culture Instructions for C-Fern® Investigations
2. Determining Sowing Density and Number of Cultures
There are approximately 1,250 C-Fern spores per mg dry weight.
Standard sowing density is approximately 300 spores per 60-mm
petri dish. Table 1 provides information for sowing spores at various
densities. Note: Depending upon technique, there is always some
spore loss during sowing procedures. Therefore, the spore
numbers given are approximate.
Table 1. Spore-sowing densities
FOR
this
density
1
USING
10- or 5mg vials
ADD
this much
water (mL)
to vial
SOW onto petri dish
standard
10
4
3
-
36
300+
standard
5
2
3
-
18
300+
2/3
10
4
2
1
54
200+
2/3
5
2
2
1
27
200+
½
10
2
3
-
18
150+
½
5
4
3
-
36
150+
1/3
10
4
1
2
108
100+
1/3
5
2
1
2
54
100+
This many drops
of spore
suspension
Plus these
additional
drops of water1
MAKE
about this
many petri
dish cultures
WITH
this number of
spores per
petri dish
Additional water is to allow enough liquid (a total of 3 drops) for spreading spores evenly on the agar surface.
3. Preparing the Culture Medium
Four options for preparing Basic C-Fern Medium are given below:
A) premade bottled medium (melt and pour)
B) & C) powdered medium (mix, autoclave or microwave, and pour)
D) preparation and use of stock solutions.
A. Using Pre-Made Bottled C-Fern Medium
Materials:
• Bottled C-Fern Medium (Basic C-Fern Medium is available in
160- and 400-mL bottles. C-Fern Medium is also provided in
most kits or kit refills.)
• Sterile Petri Dishes, 60 × 15 mm (Petri dishes are also provided
in most kits or kit refills.)
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Culture Instructions for C-Fern® Investigations
• Hot Water Bath
• Clean Area for Pouring Dishes (or a sterile bench or laminar
flow hood)
Melt and pour medium into dishes in advance to allow sufficient
time for medium to cool to room temperature and solidify
completely. To speed melting, vigorously shake the bottle to break
up the medium before placing the bottle in a hot water bath. To melt
the medium, loosen the cap and place the bottle into a hot water
bath such that the water level is only just above the level of the
medium in the bottle. Do not fully submerge the bottle. A cover on
the water bath helps the medium to melt faster. In a boiling water
bath (100°C or 212°F), the melting time for medium is about 15
minutes for 160 mL and 45 minutes for 400 mL.
Medium should be poured in a clean area free from drafts and
traffic. Basic C-Fern Medium lacks a carbohydrate source (sugars),
so contamination problems, if any, are usually minimal if directions
and precautions are followed. Prepare the area by wiping it down
with 70% ethanol, 70% isopropanol, or a damp, clean sponge.
Open a sleeve of 60 × 15-mm petri dishes by cutting through the
end of the sleeve with scissors. Save the sleeve for storage of
prepared dishes. Gently swirl the medium in the bottle to be sure
that it is thoroughly mixed and completely melted. Remove the
bottle cap, then tilt the lid of a petri dish upward just enough to
permit pouring of the medium into the dish. Fill dishes about 3/4 full,
i.e., 15 mL for 60 × 15-mm petri dishes; 160 mL of medium should
prepare about 10 petri dishes, and 400 mL about 25 dishes. Do not
under-fill dishes. Sufficient medium is needed for proper growth and
development of C-Fern through the sporophyte stage. Replace the
petri dish lid and allow dishes to cool undisturbed. Condensation
that may form on petri dish lids during cooling is minimized if dishes
are poured and cooled in stacks of 5–10 dishes. Once dishes have
cooled and the medium solidified, they may be returned to the
plastic sleeves, sealed with tape and stored at room temperature
for several weeks prior to use in an investigation. Unused dishes
may be stored in sleeves in a refrigerator for several months, but do
not freeze them.
B. Using Powdered Basic C-Fern Medium
Materials:
• Powdered Basic C-Fern Medium
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Culture Instructions for C-Fern® Investigations
Table 3. Composition of basic C-Fern medium stock solutions and final medium
Nutrient Components
Stock
Solution (g/L)
Final Medium (mL
Stock/L)
Final Medium
Composition
(mg/L)
100
1 10× Macronutrients
NH4NO3
1.25
125
KH2PO4
5.00
500
MgSO4•7H2O
1.20
120
CaCl2•2H2O
0.26
26
5
2 200× Micronutrients
MnSO4•H2O
0.0500
0.25
CuSO4•5H2O
0.0740
0.37
ZnSO4•7H2O
0.1040
0.52
H3BO3
0.3720
1.86
(NH4)6Mo7O24•4 H2O
0.0074
0.037
10
3 100× Chelated Iron Solution
FeSO4•7H2O
2.78
27.8
Disodium EDTA•2H2O
3.73
37.3
a
This formulation is based on a medium described in Klekowski, 1969 (Klekowski. 1969. Botanical
Journal of the Linnean Society 62: 361-377). Higher concentrations of macronutrients in the stock
solution are unstable and may form precipitates, as will most combinations of macronutrient,
micronutrient, and chelated iron stock solutions.
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Culture Instructions for C-Fern® Investigations
•
•
•
•
•
•
•
•
•
1-L Volumetric Flask or Graduated Cylinder
Distilled or Deionized Water
2-liter Erlenmeyer Flask(s) (per 1 liter medium)
Bacto Agar
Magnetic Stir Plate and Magnetic Stir Bar
pH Meter
1 M NaOH Solution
Autoclave or Microwave
Sterile Petri Dishes, 60 × 15 mm
To prepare 1 L of Basic C-Fern Medium, open the packet of
powdered medium and add it to 800 mL distilled water in a
volumetric flask or graduated cylinder. Rinse out the powder
remaining in the packet with distilled water and bring the medium to
a final volume of 1 L. For agar-solidified medium, transfer the
nutrient solution to a 2-L Erlenmeyer flask and add 10 g (1% w/v) of
Bacto Agar (Difco Laboratories). Note: Some plant tissue culturegrade agars and agar substitutes can result in inhibited or abnormal
growth of gametophytes or sporophytes and should, therefore, be
avoided. Adjust the nutrient medium to pH 6.0 using 1 M NaOH.
Cover the top of the flask with foil and autoclave the nutrient
medium at 120°C/20 psi for 15 minutes, or see the protocol that
follows for using a microwave.
Some of the powdered nutrients or agar may not completely
dissolve even after autoclaving or microwaving. This is not a
problem. Dispense medium into petri dishes, i.e., about 15 mL in a
60- × 15-mm dish and 40 mL in a 100- × 15-mm dish. The dishes
should be about w full. This ensures an adequate nutrient and
water supply through to the young sporophyte stage. One liter of
nutrient medium should pour about 55 60- × 15-mm dishes and 20
100- × 15-mm dishes. Allow the dishes to cool, completely
undisturbed. Condensation on petri dish lids is minimized if the
dishes are poured and cooled in stacks. Once the dishes have
cooled and the medium has solidified, they may be returned to the
plastic sleeves, sealed with tape, and stored at room temperature
prior to use in an investigation. Unused dishes may be stored in
sleeves in a refrigerator for several months. Do not freeze.
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Culture Instructions for C-Fern® Investigations
C. Using a Microwave to Prepare C-Fern Powdered
Medium
If an autoclave is not available, a microwave can be used to
prepare Basic C-Fern Powdered Medium. Although the lower
temperatures and shorter times of microwaving cannot guarantee
sterility to the degree that autoclaving does, this method has been
used repeatedly to successfully prepare contaminant-free medium.
Use caution when heating and handling! Wear safety glasses, use
gloves, and do not leave the microwave unit unattended!
To prepare 1 L of Basic C-Fern Medium, open the packet of
powdered medium and add it to 800 mL of distilled water in a
volumetric flask or graduated cylinder. Rinse out the powder
remaining in the packet with distilled water and bring the medium to
a final volume of 1 L. For agar-solidified medium, transfer the
nutrient solution to a 2-L Erlenmeyer flask, then add 10 g (1% w/v)
Bacto Agar (Difco Laboratories). Note: Some plant tissue culture
grade agars and agar substitutes can result in inhibited or
abnormal growth of gametophytes or sporophytes and should
therefore be avoided. Adjust the nutrient medium to pH 6.0 using
1 M NaOH. Cover the top of the flask with plastic wrap and process
the solution in a microwave unit as specified in Table 2. You may
need to compensate for microwave units of different powers by
adjusting the suggested times. Plates should be poured 3/4 full.
Table 2. Steps for using a microwave to prepare 1 L of medium1
Step
Microwave Time
(1000-W output unit)
State of Solution2
Post-Microwave
Procedure3
1
5 minutes
hot
remove, swirl
2
1 minute
boiling
remove, swirl
3
15 seconds
boiling
remove, swirl
4
15 seconds
boiling
remove, swirl
5
15 seconds
boiling
swirl to mix and pour petri dishes
1
Be sure to use a vessel at least twice the volume of the medium.
2
The solution will boil vigorously in Steps 2–5. The medium must be visually monitored constantly so the power can
be reduced or turned off briefly if it starts to boil over.
3
It is important to mix the medium thoroughly by swirling the flask after each step and while pouring the plates.
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Culture Instructions for C-Fern® Investigations
D. Preparation of Basic C-Fern Medium From Stock
Solutions
Materials (Preparation of Stock Solutions):
• Macronutrient, Micronutrient, and Fe Salts (refer to Table 3 for
list)
• Distilled or Deionized Water
• 1-L Volumetric Flask(s) or Graduated Cylinder
• Microbalance
• Magnetic Stir Plate and Magnetic Stir Bar(s)
• For Fe Stock Solution:
• Hot plate,
• 2-L Erlenmeyer Flask(s),
• Watch Glass
• Storage bottle(s)
All components of Basic C-Fern Medium should be prepared using
high quality distilled and/or deionized water.
Prepare macronutrient stock solution and micronutrient stock
solutions separately by dissolving all listed quantities of
components (see Table 3) individually, in sequence, into about 800
mL of distilled water; bring to a 1liter final volume. Both
macronutrient and micronutrient stock solutions can be autoclaved.
Autoclaved stock solutions will keep for over 6 months and should
be stored in glass at 4°C.
Prepare Chelated Fe-EDTA stock solution by dissolving each
component separately in ca. 450 mL of water. On a hot plate, heat
the EDTA solution to boiling and then add the hot EDTA solution
TO the FeSO4 solution. Cover with a watch glass and boil
combined solutions for 1 hour, cool completely, then bring to 1-L
volume. Store Chelated Fe-EDTA solution in glass at 4°C.
Materials (Preparation of Final Medium):
•
•
•
•
•
•
•
Macronutrient, Micronutrient, and Fe Stock Solutions
100-mL Graduated Cylinder
10-mL Pipet
1-L Volumetric Flask or Graduated Cylinder
Distilled or Deionized Water
2-L Erlenmeyer Flask (per 1 liter of medium)
Bacto Agar
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Culture Instructions for C-Fern® Investigations
•
•
•
•
•
•
pH Meter
Magnetic Stir Plate and Magnetic Stir Bar
1 M NaOH Solution
Foil (to cover flask during autoclaving)
Autoclave
Sterile Petri Dishes, 60 × 15 mm
To prepare Basic C-Fern Medium, add the appropriate volume of
each of the three stock solutions to about 800 mL of distilled water
in a volumetric flask or graduated cylinder and bring to 1-L final
volume. For agar-solidified medium, transfer nutrient solution to a
2-L Erlenmeyer flask, add 10 g (1% w/v) agar (Bacto Agar). NOTE:
Some plant tissue culture grade agars and agar substitutes can
result in inhibited or abnormal growth of gametophytes or
sporophytes and should therefore be avoided. Adjust nutrient
medium to pH 6.0 using 1 M NaOH. Autoclave nutrient medium at
120°C/20 psi for 15 minutes. Dispense medium to petri dishes.
Dishes should be about w full—about 15 mL in a 60- × 15-mm dish
and 40 mL in a 100-mm dish. This ensures an adequate nutrient
and water supply through to the young sporophyte stage. One liter
of nutrient medium should pour about 55 60-mm dishes and 20
100-mm dishes. Allow the dishes to cool, completely undisturbed.
Condensation on petri dish lids is minimized if dishes are poured
and cooled in stacks. Once the dishes have cooled and the medium
solidified, they may be returned to the plastic sleeves, sealed with
tape, and stored at room temperature prior to use in an
investigation. Unused dishes may be stored in sleeves in a
refrigerator for several months. Do not freeze.
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Culture Instructions for C-Fern® Investigations
Seven Good Habits for Sowing C-Fern Cultures
1. Work very carefully when sowing spores. Secure the vial in a
convenient holder.
2. Suspend spores before every sowing.
3. Immediately sow three uniform drops onto the agar surface. Hold
pipet at a constant angle.
4. Do not touch the pipet to the agar surface.
5. In most Investigations, use a sterile spore spreader to distribute
spores uniformly over the agar surface.
6. Keep the petri dish lid in place as much as possible.
7. Label dishes with name or initials, the date spores are sown, and
the treatment code, if any.
4. Inoculating (Sowing) Cultures Using Presterilized CFern Spores
Materials:
• Surface Sterilized C-Fern Spores in a Graduated Spore Vial
(appropriate pre-sterilized spores are provided in most kits or
refills; spores for wildtype and mutant stocks are also available
separately.)
• Sterile Distilled Water (provided in most kits or refills)
• Sterile Transfer Pipet (plastic; provided in most kits or refills)
• Sterile Spore Spreader (metal spreader [e.g., a paperclip bent
into a T-pin shape] or alternative provided in most kits)
• Petri Dishes Containing C-Fern Medium (prepared in advance)
Preparing Spores Before opening any spore vial, be sure that all
spores are at the bottom of the vial by tapping the bottom of the vial
on a hard surface. Transfer the appropriate amount of sterile
distilled water to the spore vial using a sterile transfer pipet. Do not
return any water to the sterile water bottle. See Table 1 for the
amount of water to add and sowing densities. Wet spores
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27
Culture Instructions for C-Fern® Investigations
completely by firmly attaching the cap and inverting the vial two or
three times. With the cap on, check the bottom of the vial to be sure
that all spores have been suspended. Allow the spores to soak for
15 minutes prior to sowing.
Sowing Spores To
achieve consistent
sowings, spores should
be thoroughly suspended
between each sowing.
Suspend spores gently by
drawing the liquid along
with the spores in and out
of the pipet. To sow,
withdraw a small amount
of the spore suspension
into the pipet and
immediately dispense 3 Figure 9.
drops—not squirts—onto
the agar surface (Figure 9). When sowing, tilt the lid of the petri
dish upward just enough to permit access of the pipet tip. Do not
touch the agar surface with the tip of the pipet. Resuspend the
spores in the vial between each sowing by gently squeezing and
releasing the pipet bulb.
Spreading Spores To
make a sterile spore
spreader, bend a paper
clip into a “T” shape, as
shown in Figure 10. While
holding the straight end,
wipe the “T” end with the
alcohol prep pad and let it
air dry.
Figure 10.
Allow the spreader to rest
on the agar surface
Figure 11.
without pressure and
move it gently back and forth across the surface of the agar while
rotating the dish slowly with the other hand (Figure 11). The goal is
to uniformly distribute spores over the entire surface of the medium.
This may require some practice but is worth the effort—evenly
spread cultures are easy to observe and work with.
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Culture Instructions for C-Fern® Investigations
5. Maintaining C-Fern Cultures
Any number of plant-growing systems can be easily adapted to
provide adequate environmental conditions for C-Fern. However, in
order to attain the growth and timing of development shown in
Figure 1, cultures require correct and controlled temperature and
adequate lighting. If your temperature and lighting conditions differ
substantially from that indicated here, a test run should be carried
out to determine when, under your conditions, specific
developmental stages will be present for observation and
manipulation. The descriptions of two options, Culture Domes and
Growth Pods, follow.
A. Culture Domes
®
Once cultures are inoculated with spores, place them into Culture
Domes consisting of clean plastic greenhouse trays covered with
transparent humidity domes. For best results, Culture Domes
should be thoroughly clean. Both trays and domes are available
from distributors of C-Fern products. Culture Domes serve to
reduce the possibility of contamination, variations in temperature
and humidity, and permit easy handling of a larger number of
dishes. Do not tightly seal the petri dishes, for example with
Parafilm, as this can result in poor growth, presumably due to
ethylene buildup.
B. Growth Pod
The C-Fern® Growth Pod™ can replace or complement the
standard Culture Dome that was recommended in early versions of
the C-Fern Manual. The pod’s reduced space requirement,
improved temperature control, and increased portability are highly
advantageous, especially in situations where the optimum culture
temperature of 28°C (82°F) is difficult to attain using the standard
Culture Dome. Growth Pods (without light) are available from
distributors of C-Fern products, or you can make them yourself, as
follows.
The C-Fern Growth Pod is made using insulated, vinyl 6-pack
coolers or student lunch boxes that are readily available
commercially. When fitted with an interior cardboard box, lined with
aluminum foil or tape and covered with a fitted lid of 1/4”clear
acrylic, the pod can be filled with six stacks of five petri dishes (60 ×
15 mm), enough for a class of 30 or more students. A simple
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29
Culture Instructions for C-Fern® Investigations
lighting fixture consisting
of a 6”reflective dome and
a switch (small clamp or
utility lights work well) and
a 15-W screw-in
fluorescent bulb is all that
is needed. The newer
screw-in fluorescent bulbs
are long lasting, highly
efficient, and feel only
warm to the touch, even
when left on continuously.
The light can be rested
directly on the acrylic lid,
upon stacks of petri
dishes or small blocks on
top of the acrylic lid, or it
can be suspended over
the top of the lid. The
height should be adjusted
to achieve a 28–30°C
(82–86°F) internal
temperature. Small,
inexpensive digital
thermometers are handy
to keep track of
temperature.
Thermometers that have
an “outside” temperature
probe on the end of a thin Figure 12. C-Fern Growth Pod
wire are especially useful.
The Growth Pod provides a bright, humid, and warm environment
for rapid gametophyte and young sporophyte development.
Because of the stacking arrangement of the petri dishes, cultures
on the bottom may develop slower than those on the top. Reversing
the order of the stacks after 6 or 7 days can minimize this.
However, the slight variation in gametophyte size that results from
the stacking can be beneficial for observations and use. For local or
longdistance transport, the top of the Growth Pod can be zipped
closed to reduce temperature and humidity fluctuations. The light is
small, portable, and can be plugged in anywhere.
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Culture Instructions for C-Fern® Investigations
C. Temperature
The optimum temperature for spore germination and gametophytic
development is about 28°C (82°F), as measured inside the Culture
Dome or Growth Pod. This is somewhat higher than for many other
plants. Similar growth and development of gametophytes can be
obtained at 26–30°C (79–86°F). However, temperatures lower than
this will substantially alter developmental timing; for example,
development will take twice as long at 20°C (68°F). It is a good idea
to monitor and record the temperature inside your Culture Dome or
Growth Pod daily. Control of the temperature inside the Culture
Dome or Growth Pod can be achieved by adjusting the distance
between the light source and Culture Dome or Growth Pod. Once a
suitable temperature is achieved, the height of the lights should
remain constant during all phases of culture.
A constant temperature within the Culture Dome or Growth Pod
reduces the chances of condensation on petri dish lids. If
condensation is a problem, cultures may be grown upside down
once the sowing water has been absorbed into the agar medium.
D. Light
Note: Continuous illumination is recommended
i. Growth Pod Requirements
A screw-in 15-W fluorescent bulb with a simple fixture, such as a
clamp or garage light, is long lasting, safe, and easy to use for light
and temperature maintenance. See the above description for
constructing Growth Pods.
ii. Culture Dome Requirements
Continuous illumination by two 40-W cool-white fluorescent tubes at
a distance of 45 cm from the cultures will accommodate two
standard Culture Domes (54 × 27 cm). This will provide about 80
µmoles of photosynthetically active radiation m–2sec–1, depending
on the age of the bulbs. Two Culture Domes provide enough space
for up to 64 individual 60- × 15-mm petri dishes. Smaller or larger
setups can be used to serve individual needs. Temperature inside
the Culture Dome is more important than light level, so the distance
between the Culture Dome and the light source should be adjusted
to obtain a temperature near the optimum.
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Culture Instructions for C-Fern® Investigations
iii. Constructing a C-Fern Light Stand
With inexpensive and
simple materials, you can
easily construct a C-Fern
light stand.
Materials and Tools:
• 10' of PVC Pipe
(1”internal diameter,
ID)
• 2 L-Shaped PVC
Connectors (1” ID)
• 2 T-Shaped PVC
Connectors (1” ID)
• 4 PVC End Caps (1”
ID)
• Tape Measure
• Hacksaw
• Marker
(The PVC pipe and
fixtures in a variety of
sizes are readily available
in most self-serve home Figure 13. Construction of C-Fern Light Stand
improvement stores.)
1. Mark the pipe to the
correct lengths, as
follows:
• 4 6” light bank “feet”
• 2 24” risers
• 1 24”or 48”
crossbeam
2. Cut the pipe with a
hacksaw or PVC pipe
cutter.
3. Construct the light
bank by securely
pushing the pipes into
the appropriate
Figure 14. Complete C-Fern light bank
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Culture Instructions for C-Fern® Investigations
connectors. Adjust the light bank “feet” so they are parallel. Hang
the light source from the crossbeam, and you are ready to grow CFern. Do not glue the PVC pipe if you want the light bank to be
portable and easy to store.
When your light stand is finished, you’ll need a light source. For CFern, use a 2-foot fluorescent fixture (4 F20-W cool-white bulbs)
OR a 4-foot fluorescent fixture (2 F40-W cool-white bulbs) OR 2 or
more dome reflector fixtures with screw-in 15-W fluorescent bulbs.
Light systems suitable for C-Fern are available from distributors of
C-Fern products. Grow your C-Fern cultures inside a Culture Dome
or Growth Pod under continuous light and at a temperature of about
28°C (82°F). Adjust temperature inside the Culture Dome or Growth
Pod by varying the distance between it and the lights.
6. Observing Cultures
Observations of germinating C-Fern spores, developing
gametophytes, and swimming sperm can be conveniently made at
low magnification (10×–40+×) using a stereomicroscope.
Illumination from below, i.e., with transmitted light, is best. Because
the stage of a microscope with transmitted illumination may
become quite warm after extended use, students should be
encouraged to turn the base light off and to remove the culture
plates from the stage when they are not being observed. If cultures
are to be observed with the petri dish lids in place, condensation
may form on the inside of the lid and obstruct observation. If spare
lids from clean unused dishes are available, these can replace the
fogged lids. If spare lids are not available, it is possible to remove
the condensation by carefully wiping the lids with a clean,
preferably sterile, tissue. If the lids are not kept in place during
observation, gametophytes may begin to show signs of drying after
being open for several minutes. In this case, it is important to
remind students to place the lids back on the cultures when the
cultures are not being observed. Observations can also be made
using a compound microscope (40×–400×) by making wet mounts
of some of the gametophytes. Select and remove gametophytes
from the culture medium with a sterile probe or toothpick.
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Culture Instructions for C-Fern® Investigations
7. Sporophyte Culture
A. Standard C-Fern (RN cultivar) Sporophytes
Although most student investigations using C-Fern concentrate on
gametophyte development and sexual reproduction through the
early sporophyte stage, 14–21 days following sowing (DFS),
extended observations of sporophyte development, including the
production of the next generation of haploid spores, are often
desirable. Under adequate conditions, sporophytes with mature
spores can be grown within 90 DFS. Because of its tropical nature,
a large-scale culture of C-Fern sporophytes requires a warm and
humid greenhouse environment. However, culture of individual
sporophytes can be easily accomplished in a terrarium or other
suitable vessel. The following instructions are provided for growing
individual sporophytes in 2-L clear plastic beverage bottles.
1. One to 3 weeks after water has been added to the gametophyte
culture to facilitate fertilization, young sporophytes should be
transferred individually to separate 60- × 15-mm petri dishes
containing Basic C-Fern Medium. Transfer can be made with a
clean toothpick or probe so that the root end of the sporophyte is
imbedded in the agar and the first leaf is above the agar. Use care
to avoid contamination.
2. Place the sporophyte cultures in the Culture Dome or Growth
Pod under the same lighting and temperature conditions that were
used for gametophyte cultures. Sporophytes should remain under
these conditions for 2–4 weeks. Usually, it is not necessary to add
water, but if the culture begins to get dry, use a pipet to add sterile
distilled water as needed.
3. When sporophytes have produced several roots and leaves, they
can be transferred to a bottle “terrarium.” If the sporophyte is small
enough, both it and the soil mix (see step 4) can be pushed through
the mouth of the bottle and adjusted/planted with a long narrow
stick, etc. Otherwise, make a circular cut 2 inches below the neck.
Cut only w of the distance around the bottle so that a “hinge” is left.
The top can now be lifted to provide a larger opening for planting.
4. Place 2 cups of pre-moistened (damp but not wet)
ProMix®Potting Soil in the bottom of the bottle. Add 4–6 mediumsized beads of Osmocote® 14-14-14 Fertilizer and mix them into the
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top 2-inch of potting mix. Make a shallow concave cavity (2"deep ×
1"diameter) in the center of the bottle.
5. Carefully remove a sporophyte from its petri dish by lifting the
agar out of the dish, roots and all. A flat knife or thin spatula works
well for this. Remove as much agar as possible without damaging
the roots and place the sporophyte (roots down!) in the cavity made
in step 4. Gently cover the roots with potting mix and lightly press
down on the surface around the sporophyte to give it a firm footing
in the potting mix.
6. Add enough distilled water to thoroughly moisten the potting mix
and help displace any air spaces between the roots and soil mix.
The mix should be moist but not soaked, although excess water will
not typically harm C-Fern. Subsequent watering can usually be
made as needed, usually on a weekly basis. Use distilled or bottled
drinking water, if available. Note: Constant moist conditions will
result in mostly vegetative growth. If fertile leaves with spores are
desired, a moderate level of water stress will typically cause a
change from vegetative to reproductive leaf production. This can be
accomplished by allowing the soil to dry substantially between
waterings.
Place the terrarium under the same lighting conditions used for the
gametophyte cultures. Warm temperatures and constant 24-hour
illumination work best; cold window sills are not recommended.
Cooler temperatures and less light will slow development but still
allow growth. Other than watering, as described in step 6, little care
is needed.
Consult the C-Fern® Web Manual section for additional information
on the greenhouse culture and manipulation of sporophytes.
B. C-Fern Express Sporophytes
C-Fern Express is a new strain of C-Fern® that has been developed
to enhance student investigations through to the fertile sporophyte
stage of the life cycle. Derived from two Japanese varieties of
Ceratopteris thalictroides, C-Fern Express exhibits very rapid
sporophyte development. It can be substituted for the RN cultivar
in investigations of the fern life cycle. Culture requirements for
gametophytes are essentially the same as the standard C-Fern
strains, with some slight developmental differences in gametophyte
morphology. The following guidelines for sporophyte culture will
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Culture Instructions for C-Fern® Investigations
allow studies of the entire life cycle, from spore to spore, within a
60-day period.
C-Fern Express sporophytes can be grown to maturity within the
same size petri dishes that are used for gametophyte culture.
Dishes should be 2/3 full with Basic C-Fern Medium. At 1-2 weeks
of age, use a sterile toothpick to transfer young sporophytes to
fresh dishes (4 per dish). Maintain the cultures in a Growth Pod or
similar container under the same conditions for gametophytes.
However, cooler temperatures as low as 75 ºF helps to accelerate
maturation, while limiting growth. As the medium dries over the
next several weeks, add a small amount of water to keep things
moist. Avoid adding excess water that cannot be absorbed by the
agar.
Monitor the sporophytes weekly and note developmental changes
in the leaves. Nutrient and space limitations speed up the change
from vegetative leaves (round to oval shaped) to fertile leaves
(becoming more finely dissected with narrow lobes). After a few
weeks, the edges of the fertile leaves will roll inward.
Subsequently, several round sporangia will be observed within and
along the length of the rolled edges (use a 10X hand lens or
microscope). The processes of spore formation from meiotic
division and spore maturation will occur over the next few weeks,
until the sporangia and spores are brown. Maturity is marked by a
distinct brown line on the underside of the leaf. At this time, spores
can be harvested by removing the leaves and drying them out in a
clean dry petri dish. Under the conditions described, total time from
first starting the cultures to harvesting mature spores is typically 60
days. See the web pages (www.c-fern.org) for images.
8. Additional Information For Educators
A. Hints for Large Classes
The 40-mg vial of unsterilized bulk spores is a convenient size for
larger classes. To sterilize a single batch or multiple batches of this
quantity of spores, it is advisable to sterilize each 40-mg quantity
using the standard techniques described in the Culture Instructions.
After sterilization has been completed, transfer the sterile spore
suspension to a sterile test tube of larger capacity (e.g., =20 mL).
Then, for each 40-mg quantity, bring the liquid volume up to 16 mL
with sterile distilled water. Dispensing 3 drops per dish from a
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Culture Instructions for C-Fern® Investigations
sterile transfer pipet will generate approximately 140 small petri
plates. Be sure to keep the spores suspended by repeatedly
drawing them in and out of the pipet during the inoculation
procedure.
All of the spore suspension can be sown at one time. However, if
laboratories are to last for more than 2 days, it may be advisable to
sow only a portion (perhaps one-half) initially. This can be
conveniently done by separating the sterilized spore suspension
into two equal volumes. One volume can be kept in the dark for 1–3
days, as is done in the DarkStart procedure. In this way, cultures of
approximately the same age can be scheduled for an entire week
of laboratories. This can even be a convenient way of bridging
schedule difficulties caused by weekends or vacation days.
B. Independent Student Research Projects
C-Fern is an excellent tool for use in independent student research.
Projects can be completely independent of the normal classroom
activities or may be integrated with group exercises. The principal
goals of independent research are to stimulate students' interest in
the subject by encouraging them to formulate questions and
subsequently to design appropriate experiments that can provide
answers to those questions. This directly involves students in the
process of science. Perhaps the most important task of the teacher
in this situation is to give helpful but minimal advice and guidance in
order to avoid a level of frustration that can dampen students’ initial
enthusiasm or willingness to undertake a project.
The individual investigations contained within the C-Fern®®Manual
can serve as a starting point to generate questions to pursue. In
particular, some of the open-ended questions that are presented at
the end of the exercises may be useful. Some of these, along with
others, follow. Another source is the bibliography contained in Part
C of the C-Fern Manual, which gives references for a number of
separate investigations that involve a variety of experimental
approaches. Use of mutagenesis and selection procedures,
determinations of the effects of various growth hormones,
comparative tests of the responses of wild type and various
mutants to environmental stresses and herbicides, and many other
examples are contained within the cited references. Emphasize to
the students that many other questions, likely the most interesting
ones, haven’t even been asked yet!
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To cultivate independence in students, it is perhaps best to provide
them with only the minimal materials that are needed for C-Fern
culture. These materials could consist of vials of unsterile spores of
the wild type or particular mutants, as well as the basic materials for
preparing nutrient medium. In this way, students gain more
familiarity with the organism by performing necessary tasks that are
often completed by the teacher or others prior to students’
involvement with structured exercises. Very good questions often
arise from these types of basic manipulations of the organism.
Environmental parameters, such as temperature and light, can be
conveniently varied by altering the distance between the Culture
Dome or Growth Pod and the light source, and/or by utilizing
various types of shading material to decrease the amount of
incident light on the cultures. Manipulation of other parameters,
such as medium composition, pH, sowing density, population
composition, light quality, day length, etc., can also be
accomplished easily.
C. Questions for Discussion
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
How far do sperm swim?
How do sperm move?
How do sperm find the archegonium?
Why are archegonia restricted to the central region of the
hermaphrodite?
Can sperm respond chemotactically to artificial (chemical)
signals?
How do sperm reach the egg after finding the archegonium?
How do gametophytes limit fertilization to 1 and rarely 2 events?
What happens to gametophytes after a fertilization event?
What happens to meristem activity after a fertilization event?
What type of signal elicited by a fertilization event is responsible
for stopping meristem activity?
What are the patterns of cell division and expansion that yield
the heart-shape of the hermaphrodite?
What are the effects of gravity on developing gametophytes?
How long will unfertilized gametophytes live?
Other than using a bioassay, what approach could you use to
show that a chemical substance was responsible for controlling
development of sexual types in C-Fern?
Do all gametophytes secrete the pheromone?
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• When are gametophytes sensitive to the pheromone?
• How could you chemically characterize the active agent?
• How could you design a bioassay to detect other agents that
control development?
• What might be the advantage of controlling the sex ratio in the
cultures?
• What other factors might be important in controlling the sex ratio
in cultures?
• How could one determine if there was a genetic difference that
controlled sexual type in gametophytes?
• How could you test the relative fitness of different genotypes,
e.g., wild type versus polka dot?
• Can you think of an advantage to having more males at higher
densities? How could you test your hypothesis?
• How do higher densities result in more males within a
population?
• What might be the advantage of having fewer males at lower
densities?
• How do changing proportions of males influence the kinds of
mating that can occur?
• •What factors limit sporophyte growth at high densities?
• Can sporophytes form without fertilization?
• Can gametophytes develop directly from sporophyte tissue?
• What role does the meristem play in maintaining the
hermaphroditic gametophyte?
• Can microsurgery on gametophytes demonstrate alternate
paths of development?
• Can sporophytes develop normally if the young embryo within
the archegonium is cut away from the gametophyte?
• What effect on development is caused by adding sucrose to the
medium?
• What factors are important in sporophyte development?
• Can sporophyte development be speeded up?
• What is the difference between vegetative and fertile leaves on
sporophytes?
• Do rhizoids respond to the direction of gravity?
• Do gametophytes show a phototropic response?
• Do sporophytes show a phototropic response?
• What wavelengths of light are necessary to induce germination?
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Culture Instructions for C-Fern® Investigations
• Does gametophyte growth and development respond to
different wavelengths of light?
• Which phase, gametophyte or sporophyte, is more important in
limiting the distribution of C-Fern?
• Under what conditions is vegetative development more
important than sexual development?
• Do rhizoids have a specific function?
• If a meristem is surgically removed, will a new one form? What
factors could influence this?
• Is distance between gametophytes important during their sexual
development?
• Do sperm swim best in distilled water or in some other medium?
• Following meiosis, how long does it take a spore to mature?
• How long can sperm swim? What environmental factors are
important in determining this?
• What happens when C-Fern cultures are grown upside down or
on their side? Why?
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Additional Culture Methods and Techniques
Additional Culture Methods and Techniques
Surface Sterilization of C-Fern Spores
The procedure outlined on the next page describes how to surface
sterilize spores that are either collected directly from sporophytes or
obtained in bulk, unsterilized lots. Before you begin,
Make sure the area is clean and free from drafts and traffic.
•Wipe down the area with 70% ethanol, 70% isopropanol, or a
damp clean sponge.
Materials:
• Bulk Unsterilized C-Fern Spores (For information on wild-type
and mutant spores, refer to the Sport Reports - Descriptions of
C-Fern Mutants section and distributors of C-Fern products.)
• Sterile Tube With Conical Bottom
• Sterile Transfer Pipets
• Sterile Distilled Water
• Plain Commercial Laundry Bleach or Equivalent (5.25% sodium
hypochlorite solution)
• Waste Container
• Timer
NOTE: For handling larger amounts of spores (15–250 mg) it may
be more convenient to use 12- to 15-mL conical centrifuge tubes.
Other materials, such as conical microfuge tubes and automatic
pipets, may also be successfully used for spore sterilization, but
should be tested first.
DarkStart Method
In some cases, it is desirable to decrease the length of time
required to obtain gametophyte material at a particular
developmental stage, or give greater control over the timing of
particular developmental stages. Because the 10–12 days required
to obtain sexually mature gametophytes under standard conditions
includes approximately 2 days of imbibition (absorption of water) for
dry spores, it is possible to “jump start” cultures by adding water to
the vial and immediately placing the sealed vial into a foil bag or
covering it with two or more layers of foil. Complete darkness will
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Additional Culture Methods and Techniques
Step-by-Step Procedure for Spore Sterilization
1. WEIGH SPORES. Weigh out the spores onto glassine weigh paper and transfer
them to a sterile conical tube. Using 4 mL of water, 10-mg spores will sow about 35
petri dishes at a density of 300+ spores per dish.
2. PRESOAK SPORES. Cover the spores with 1–2 mL of distilled water. The
spores may be soaked just long enough to become wet, i.e., about 5 minutes, or
for up to 24 hours. To hasten the wetting of spores, tightly seal the vial and invert it
2–3 times.
3. REMOVE PRESOAK WATER. Insert the sterile pipet into a conical tube and
suspend the spores by bubbling a small amount of air into the water (see Figure 4).
While air is slowly bubbling out of the pipet, gently but securely seat the pipet onto
the base of the conical tube. Sometimes it helps to gently rotate the pipet tip to
seat it properly. Squeeze the bulb to force additional air out of the pipet. When the
bulb is released, water should enter the pipet, and the spores should collect around
the outside of the base of the pipet tip, provided the pipet is securely seated on the
tube bottom (see Figure 4). If you cannot remove the liquid without bringing the
spores along, try another pipet. Remember that timing is critical when the sterilizing
solution is in the tube! Practice this technique with the presoak water prior to
sterilizing the spores. With practice, you should be able to remove the liquid, free of
spores, in about 10 seconds or less.
4. SURFACE STERILIZE SPORES. To sterilize, suspend spores in 1–2 mL of
0.875% sodium hypochlorite. Prepare 0.875% sodium hypochlorite by mixing 1
part commercial bleach (5.25%) to 5 parts distilled water. Rinse down the lip and
sides of the vial with bleach solution. To ensure that the spore mass becomes
evenly suspended, bubble air through a clean, sterile pipet. Surface sterilize the
spores for 3 minutes. Remove the bleach solution with a clean, sterile pipet using
the technique described in step 3.
5. RINSE SPORES. To rinse the spores, add about 2 mL of sterile distilled water.
The pipet used to add the rinse water may be used repeatedly as long as care is
taken to prevent contamination with foreign spores. Remove the rinse water with a
clean, sterile pipet. Repeat the rinse step 1 or 2 more times. The pipet used to
remove the sterile distilled water may be left in the tube for use in sowing spores.
6. SOWING AND SPREADING SPORES.For 10 mg of spores, add 4 mL of sterile
distilled water and proceed to the sowing and spreading of spores as outlined in
Section 4, Inoculating (Sowing) Cultures Using Presterilized C-Fern Spores. Adjust
for the density of spores per dish and the number of dishes sown as needed (see
Table 1).
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Figure 15. Liquid removal from C-Fern spore suspension.A. Spores soaking in
liquid in vialB. Pipet enters liquid while air is expelled. Bubbling before “seating”
pipet on bottom prevents spores from entering pipet. Expelling air should be
completed while pipet is seated on bottom - enough to subsequently achieve
complete aspiration of liquid.C-E. When pipet is seated on bottom, Bulb is
released to initiate aspiration. Aspiration of liquid occurs while spores remain
behind. F. Spores remain in bottom of vial.
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Additional Culture Methods and Techniques
prevent spore germination in most genotypes (the dark germinator
stock, dkg is the exception) while still allowing normal imbibition.
After 3–7 days in darkness, when spores are sown and placed
under lights, germination will occur about 2 days earlier than with
the standard light sowing technique.
DarkStart can be used to pre-start a lab so that students sow fully
imbibed spores and can more easily observe and/or measure all of
the early stages of development. For example, spores
“DarkStarted” on a Friday, Monday, or Tuesday could be sown and
exposed to light the following Friday to ensure initial germination by
Monday and complete germination by Wednesday. DarkStart also
increases synchrony of germination in cultures and reduces the
frequency of male gametophytes.
Dry Sowing of Presterilized C-Fern Spores
As an alternative to using a liquid suspension of spores for
inoculating petri dishes, spores can be sown with a sterile cotton
swab. Gently touch the tip of the swab to dry spores in the bottom
of the vial. Disperse onto the culture medium by gently tapping the
stem of the swab with a finger. Although this method does not
produce a uniform distribution or density, it can be useful to quickly
establish a few cultures without using the entire vial of spores.
Liquid Culture of C-Fern Gametophytes
For liquid culture of gametophytes, use the same conditions of
temperature and light as with the agar-solidified culture medium.
Inoculate surface-sterilized spores into sterile medium without agar
at a rate of 3–10 mg per 100 mL and cap the flask securely with
foil. In general, the flask size should be about twice the volume of
medium used, e.g., 500 mL of medium in a 1-L Erlenmeyer flask.
To reduce adhesion of spores and of very young gametophytes to
the walls of the culture flask, allow cultures to develop without
shaking for 7 days after inoculation. After 7 days, place cultures on
a shaker at 50–100 rpm, depending on the volume of solution. If the
cultures are not shaken, they will release sperm and rapidly fertilize
as soon as gametophytes become sexually mature.
Mutagenesis of C-Fern Spores
Large numbers of C-Fern spores are readily mutagenized by
chemicals (e.g., EMS) or X-irradiation as a means to enhance
mutation frequency for screening or selection protocols. For
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Additional Culture Methods and Techniques
environmental- or chemical-based selections or screenings, the
response of unmutagenized C-Fern should be thoroughly tested.
Complete dose response curves should be used to identify the
appropriate selection conditions that permit clear discrimination of a
mutant from the wild-type response. To identify visible mutations
(e.g. pigmentation, morphology), a thorough understanding of the
variation in gametophytic development is necessary.
With lethal or near-lethal selection conditions, e.g., with the
herbicide paraquat, mutagenized spores may be sown at a density
of 10 mg spores (or greater) per large petri dish (100 × 20 mm).
With nonlethal selection conditions, sow at a rate of 5 mg spores
(or less) per large petri dish to permit clear observation of individual
gametophytes.
1. Ethyl Methane Sulfonate (EMS) Mutagenesis
To initiate ethyl methane sulfonate mutagenesis, you will need the
following: C-Fern Spores 200 mM EMS (ethyl methane sulfonate)
To prepare 1 mL of 200 mM EMS solution:
21.7 µL EMS (MW=124.2, d=1.1452, 1 M=108.5 mL/L)
978.3 µL 100 mM Phosphate Buffer, pH 7.00
To make 200 mL of 100 mM Phosphate Buffer, pH 7.00:
39 mL 200 mM NaH2PO4•H2O (2.76 g/100 mL)
61 mL 200 mM Na2HPO4 (2.84 g/100 mL)
100 mL Distilled Water
0.4M Sodium Thiosulfate (Na2S2O3)
Sterile Conical Tubes or Vials (e.g., glass centrifuge tubes with a
uniform,conical inside bottom—either autoclaved or foil covered
and dry sterilized at 120°C for 2 hr; plastic, disposable centrifuge
tubes; or plastic, conical vials)
Sterile Pipets (e.g., sterile plastic pipets or autoclaved glass pipets
with sterile, rubber bulbs)
Other Supplies for Surface Sterilization of Spores (refer to the
section on Surface Sterilization of C-Fern Spores in the Culture
Instructions)
Protective Clothing and Materials Fume Hood
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Additional Culture Methods and Techniques
Caution: EMS is a potent mutagen and suspected carcinogen! Use
proper protective clothing, eye protection, gloves, and containment
tray. Carry out all mutagenesis procedures in a properly functioning
and safe fume hood according to your local institutional safety
requirements and regulations. Sodium thiosulfate has been used to
neutralize EMS spills, treatment solutions, and contaminated
glassware after mutagenesis procedures. Use an excess volume of
0.4 M sodium thiosulfate to assist in neutralization of EMS. Dispose
of unneutralized and neutralized EMS waste in a manner consistent
with appropriate institutional safety regulations and requirements.
Procedures
1. Weigh out C-Fern spores to be mutagenized and transfer them
to a conical centrifuge tube. Treat up to 250 mg of spores per tube.
2. Prepare EMS-Buffer solution fresh. Mix appropriate amounts of
buffer and EMS, and vortex very thoroughly and carefully. Note:
Because EMS is difficult to dissolve in buffer, it is very important to
vortex the mixture exhaustively. If EMS solution is properly mixed,
spores should mostly sink to the bottom after wetting and settling. If
spores are mostly floating, mixing was not adequate.
3. Add EMS-Buffer solution to completely cover spores. Treat
spores for 36 hr at room temperature.
4. Remove EMS solution and wash spores three times with about 2
mL sterile distilled water, using the technique described in the
Surface Sterilization of C-Fern Spores section. Neutralize all liquid
waste and glassware surfaces with 0.4 M sodium thiosulfate
solution.
5. Surface sterilize spores according to procedures in the Surface
Sterilization of C-Fern Spores section. Inoculate mutagenized
spores according to directions below.
2. X-Ray Mutagenesis
To initiate X-ray mutagenesis, you will need the following:
Materials:
• C-Fern spores
• Sterile Conical Tubes or Vials (e.g., glass centrifuge tubes with
a uniform,conical inside bottom—either autoclaved or foil
covered and dry sterilized at 120°C for 2 hr; plastic, disposable
centrifuge tubes; or plastic, conical vials)
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Additional Culture Methods and Techniques
• Sterile Pipets (e.g., autoclaved, glass, disposable Pasteur pipets
with sterile, rubber bulbs; sterile plastic pipets)
• Sterile Distilled Water
• Sterile Glass Petri Dish (100 × 20 mm)
• Plastic Sandwich Bag or Parafilm
• X-Ray Machine
Procedures
1. Weigh out C-Fern spores to be mutagenized and transfer them
to a conical centrifuge tube. Up to 250 mg of spores may be treated
per tube. Add sterile distilled water and soak spores for 24 hr prior
to sterilization and irradiation.
2. Surface sterilize spores according to Surface Sterilization of CFern Spores in the Culture Instructions. Using a minimal amount of
sterile water, transfer surface-sterilized spores to a sterile glass
petri dish. Seal dish with Parafilm or place dish inside a plastic
sandwich bag. 3. Irradiate spores with 25,000 Roentgens or about
555 Roentgens min–1for 45 min. After irradiation, sow spores as
usual.
After these X-irradiation conditions, spore germination at 10 days
following sowing should be about 80% of the control. A screen with
10–5 M fluorodeoxyuridine gives a mutant frequency of about 2 ×
10–5 .
Controlled Fertilization: Selfing and Crossing
Techniques
1. Self-Fertilization
Individual hermaphroditic gametophytes have both archegonia and
antheridia and can readily self-fertilize under the appropriate
conditions. To insure selffertilization and eliminate the possibility of
cross-fertilization, individual spores or sexually immature
gametophytes should be transferred to petri dishes (isolated) and
cultured until sexually mature, i.e. 10–12 days following sowing.
Use a stereomicroscope and sterile dissecting needle for the
transfers. To accomplish self-fertilization, add 0.1–0.5 mL sterile
distilled water directly on top of the sexually mature hermaphrodite.
Within a few minutes, if antheridia are fully-developed,
spermatozoids should be observed swimming and swarming
around the area of the notch meristem where mature archegonia
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Additional Culture Methods and Techniques
are found. If gametophytes are floating in the water, they can be
submerged with a sterile dissecting needle to ensure that the
archegonia are covered with water. After 3–4 days in culture,
swollen archegonia containing young embryonic sporophytes will
be observable—typically one, sometimes two, per gametophyte. If
some isolate cultures do not contain embryos, they can be
rewatered. Root and leaf formation will be evident within 1 week
after embryos are observed. Maintain cultures until the sporophytes
are large enough to be transferred to the greenhouse or a terrarium
(e.g., bottle culture).
2. Cross-Fertilization
Accomplishing cross-fertilizations or hybridizations between specific
stocks is easy if you follow procedures carefully and take care with
regard to gametophyte age and developmental stage. Cultures to
be used as male stocks should be 10–18 days DFS (under
standard culture conditions). At this age, the male stock should
contain well-defined tongue-shaped (spatulate) males with
numerous antheridia. The female stock should be 8–12 DFS (under
standard culture conditions) and contain heart-shaped (cordate)
gametophytes with one to three archegonia and few, if any, mature
antheridia. There is a very brief stage of development at which
there are 1–2 mature archegonia and no mature antheridia, so the
cordate gametophytes are effectively female. Careful attention to
gametophytic development age ensures high spermatozoid number
and activity for the male parent and 1–2 receptive archegonia for
the female with a minimal chance of selfing. In addition, all
manipulations should be carried out in a reasonably rapid and
efficient way.
Set up females for crosses first. (Removing cultures from a warm
culture room to a cooler area can result in premature release of
sperm and establishment of archegonial receptivity.) Set up isolate
cultures of the female stock by transferring individuals at the
appropriate developmental stage to separate petri dishes. Use a
stereomicroscope and sterile dissecting needle for the transfers.
Place the gametophytes on the agar surface with their archegonia
facing up. It is also a good idea not to transfer any females that are
in contact with males on the stock dish.
Once all females are transferred, add 1–2 pipets of sterile distilled
water to cover the gametophytes on the male stock culture dish.
With a stereomicroscope, monitor sperm release. Maximum sperm
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Additional Culture Methods and Techniques
activity should be evident within 5 min. With a sterile transfer pipet,
carefully collect the sperm suspension and place 1–2 drops of it on
each female to be crossed. (Avoid picking up gametophytes and
other debris.) Use a microscope to check all attempted crosses. If
female gametophytes are floating in the water, they can be
submerged with a sterile dissecting needle to ensure that the
archegonia are covered with water. Remove any males that were
carried in with the sperm suspension. Sperm should be seen
swarming around receptive archegonia. It is a good practice to
discard any attempted crosses that do not show immediate
swarming of sperm since these may subsequently self-fertilize.
At 3–4 days following the crossing attempts, check the females for
successful fertilizations as evidenced by obvious swollen regions
(embryos) at the base of the archegonia. Discard all females that
do not carry embryos. If the gametophytes, techniques, and
conditions used for crosses are ideal, 90–95% of crossing attempts
can be successful.
Maintain cultures until the sporophytes are large enough to be
transferred to the greenhouse or a terrarium (e.g., bottle culture).
Crosses must be confirmed by segregation from the F1sporophyte
or by use of a marker stock.
Preparation of Semipermanent Slides for Observation
and Analysis of C-Fern Gametophytes
Semipermanent slides of gametophytes suitable for later
observations or archival documentation can be prepared by
mounting gametophytes in a Hoyer’s/Acetocarmine mixture.
Hoyer’s medium clears and preserves tissue; acetocarmine is one
of the few stains stable in the presence of Hoyer’s medium.
To prepare the slides, you will need the following:
Materials:
•
•
•
•
•
•
Hoyer’s Medium
0.5% Acetocarmine Stain
Plastic Dropper Bottle
Slides
Coverslips
Source of Iron Oxide or Rust (e.g., a rusty nail)
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Additional Culture Methods and Techniques
Prepare mounting mixture fresh by mixing 4 parts Hoyer’s medium
and 1 part acetocarmine stain (1 mL of this mixture should be
sufficient for 7–8 slides, using 22- × 22-mm coverslips). For the
acetocarmine to stain properly, add a small amount of iron oxide to
the Hoyer’s/acetocarmine mixture. Do this by dipping an old rusty
dissecting tool (e.g., forceps) or a rusty, iron nail into the mixture
until a sufficient amount of iron oxide has leached off. Experiment
some to determine the correct amount of iron—too little gives very
weak staining, but too much yields excessive precipitation and a
strongly colored background.
Place 4–10 drops of the Hoyer’s/acetocarmine mixture onto a slide.
(For best results, be sure slides are very clean.) Transfer
gametophytes from the culture dish to the mixture. Submerge
gametophytes in the mixture. Put coverslip in place. If the
gametophytes are large or convoluted, it may be difficult to avoid
trapping some air bubbles. Place completed slides on a tray to dry
undisturbed for several days. Full clearing and staining of
gametophytes may take more than several days. Retain the
remaining mixture to add to those slides that dry out excessively.
Slides prepared with a sufficient amount of Hoyer’s/acetocarmine
can keep for well over 10 years.
To prepare acetocarmine, you will need the following:
• 45% Glacial Acetic Acid 100 mL
• Carmine 0.5 g
In a fume hood, boil acetic acid and carmine gently for 5 min in a
beaker covered with a watch glass. Shake the mixture occasionally
as it cools to room temperature. Filter cooled solution through #2
Whatman paper. Filtration takes a long time, and several changes
of paper are usually necessary. Store acetocarmine stain in brown
glass at room temperature.
To prepare Hoyer’s Medium, you will need the following:
•
•
•
•
Distilled Water 50 mL
Gum Arabic (acacia) 30 g
Choral Hydrate 200 g
Glycerin 16 mL
Dissolve gum arabic completely in the distilled water. Then, and
only then, add and completely dissolve the choral hydrate. Then
add glycerin and mix well. Medium may be diluted as needed with
small amounts of distilled water. Before using Hoyer’s medium, let it
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Additional Culture Methods and Techniques
stand undisturbed for several days in order to clarify. Store mixture
at 4°C. Caution: Choral hydrate is used as a hypnotic and sedative.
Abuse may lead to habituation or addiction. Some individuals are
extremely sensitive to the vapors of Hoyer’s medium and develop
headaches or other symptoms.
Greenhouse Culture of C-Fern Sporophytes
To set up greenhouse cultures of C-Fern sporophytes, you will
need the following: Greenhouse Containers, Cubes, or Pots
Commercial Potting Soil, or Mixture:
Pro-Mix Potting Soil (15-9705) Potting Mixture (2 parts peat moss,
1 part vermiculite, and 1 part
perlite). Adjust to about pH 6.5 with 100 g agricultural lime per 10
gal of potting mixture.
Newly transplanted sporophytes should be covered with a clean
culture dome or inverted petri dish lid for 3–5 days.
1. Transplantation
Young sporophytes grown from agar culture can be transplanted to
the greenhouse when several well-developed roots and about 10
leaves are evident. The mineral nutrient medium used for
gametophytes will not support long-term sporophytic growth.
Subculturing very young sporophytes into fresh nutrient medium
may be necessary to generate sporophytes that are large enough
for successful transfer to the greenhouse. For initial transplanting,
transfer sporophytes to small potting containers (cubes, etc.) that
contain moist Pro-Mix Potting Soil or a potting mixture. ProMix®gives excellent results when used as is. Other potting mixtures
should be adjusted to pH 6.5 as indicated above.
The potting containers can be placed in a greenhouse tray
containing 1–2 inches of water. When roots begin to emerge from
the holes in the cube, the sporophyte should be transplanted to a 6to-8-in pot. Soil should be kept moist-wet at all times; an automated
watering system helps. At all stages of growth, Ceratopteris
sporophytes require warm temperatures and high humidity; it’s a
tropical fern!
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Additional Culture Methods and Techniques
2. Fertilization
A low rate of fertilization is required for Ceratopteris and most other
ferns. We achieve good growth and fertile frond production with
PETERS General Purpose Fertilizer (20-20-20 N-P-K). A solution of
9.6 g/10 gallons should be applied once weekly by saturating the
pots or other containers. A regular watering schedule should also
be maintained so that the pots do not dry out.
Cloning of C-Fern Sporophytes
Ceratopteris produces buds on the margins of fronds. These can be
readily cultured to maintain specific stocks for spore production or
for use as experimental tissue. Larger buds that have developed
into small plantlets can be pinched from the mother plant and
transferred to small potting cubes containing ProMix. Maintain the
buds on a standard greenhouse mist bench, or alternatively, keep
them moist and covered. Once the root system is well developed
(usually about 14 days), the buds can be directly transplanted to
potting mix as described previously. Smaller buds (<1 cm) can be
surface sterilized and temporarily cultured on agar medium until
they reach adequate size for greenhouse transplantation, as
described below.
To clone sporophytes from buds, you will need the following:
•
•
•
•
•
•
•
Sterile 50-mL Plastic Centrifuge Tubes
Sterile Pipets
Sterile Distilled Water
Sterile Forceps
Household Bleach (5.25% sodium hypochlorite)
Waste Container
Container for Floating Buds (e.g., cup with plastic sandwich bag
cover)
• Culture Dishes with Basic C-Fern Medium
Procedures
Collect and Prepare Buds. Collect smaller marginal frond buds from
greenhouse plants. Buds should be no larger than about 1 cm and
most of the leaf tissue from the mother plant should be trimmed into
the shape of a small triangle. In addition, leaf tissue from the bud
should be removed. As buds are collected, float them in water in a
container covered with a plastic sandwich bag.
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Additional Culture Methods and Techniques
Sterilize and Prepare Buds. Prepare sterilization solution (1 part
household bleach to 4 parts distilled water). Carefully transfer buds
to the bottom of a sterile 50-mL centrifuge tube—do not touch sides
of tubes. Do not sterilize more than about 50 buds at one time. With
a sterile pipet add sufficient sterilizing solution to completely cover
buds. Shake tube for 1 min to thoroughly wet buds with solution.
Carefully decant sterilizing solution to the waste container; use the
sterile cap of the centrifuge tube to retain buds. Rinse and decant
buds three times with about 25 mL of sterile distilled water.
Culture Buds. Wash buds into a petri dish using sterile distilled
water. Plant buds to the medium with forceps. Push the root end of
the bud slightly into the agar to establish good contact. If small
buds are cloned, be sure that the triangle of mother sporophyte
tissue is parallel to the agar surface. Once young sporophytes have
a well-developed root system and 10 leaves, they can be
transplanted to the greenhouse or a terrarium (e.g., bottle culture).
Collection of C-Fern Spores
Sporophytes produce a heteromorphic sequence of increasingly
filiform fronds characterized by an inrolled margin or false indusium
that covers developing sporangia. When this inrolled margin
becomes brown (brown-stripe stage), the spores have developed
sufficiently for harvest.
To harvest spores, you will need the following:
• Scissors,
• Razor Blades, or Scalpel Blades
• 70% Alcohol, or Bunsen Burner or Alcohol Lamp (to sterilize
utensils)
• Collection Vessels (e.g., glassine envelopes, petri dishes,
plastic tubs)
• Sieves—U.S.A. Standard Testing Sieves No. 100 and 170 (with
openings of 150 and 90 µm, respectively)
• Collection Pan
• Storage Vessels (clean and dry—e.g., glass jars, screw-cap
plastic tubes)
• 25-mL Glass Graduated
• Cylinder
• Forceps
Procedures
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Additional Culture Methods and Techniques
For harvesting spores from individual plants, clip the mature fertile
fronds and place (stuff!) them into a labeled glassine envelope.
Various sizes of glassine envelopes can be obtained from
photographic supply houses. The envelopes should be well sealed
to prevent leakage of spores around the seams. Other collection
vessels such as large plastic petri dishes may also be used.
Between collections, utensils should be sterilized with alcohol or
flame to prevent cross contamination of genotypes.
For harvesting mass collections of spores, as from many clones of
the same genotype, clip mature fertile fronds and place them in a
large plastic tub. It is best to use a tub that can be easily sterilized,
such as by autoclaving, to prevent cross-contamination of
genotypes.
Dry material at about room temperature for about 48 hr or longer,
depending on humidity. When the material is sufficiently dry, it may
be stored indefinitely at room temperature; i.e., for at least several
years. Refrigeration is not recommended.
Small quantities of spores can be decanted directly from glassine
envelopes while retaining sporophytic material. Gentle agitation can
help release additional spores. For larger collections, spores may
be processed by sieving through (in order) No. 40 and No. 60
sieves into a collection pan. These sieves have 425- and 250-µm
openings, respectively. Small batches of dried fertile fronds are
placed onto a No. 40 sieve and lightly ground with the base of a 25mL graduated cylinder. This grinding action should release nearly
all of the spores by breaking up the fertile frond material without
completely pulverizing it. The spores should fall through the sieves
to the collection pan while the fertile frond material is retained. An
additional pass through a No. 100 sieve (150-µm opening) may
help purify spores that are heavily contaminated with pulverized
fertile frond material. Store dry spores at room temperature in the
dark in screw-cap containers.
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Sport Reports - Descriptions of C-Fern Mutants
Sport Reports - Descriptions of C-Fern Mutants
The term sport is sometimes used to refer to unusual forms of
plants or animals that typically arise by mutation. This section of the
manual includes a short descriptions, suggestions for culture and
uses and references for a variety of C-Fern mutant lines.
Mutant: polka dot; gene symbol, cp
This is a very striking visual mutant that exhibits a distinct green
polka-dot appearance in cells of both gametophytes and
homozygous sporophytes when viewed with a low-power
microscope. In sporophytes, older leaves of individuals
homozygous for this mutation have an attractive silver-green
appearance. Although there is a slight reduction in growth and a
decrease in spore viability in spores produced from homozygotes,
gametophytes carrying the polka-dot mutant and homozygous
polka-dot sporophytes grow nearly as well as the wild type. This
recessive trait has been observed in several independent
selections that have been generated using both X-rays and the
chemical mutagen EMS. A study of the trait, using transmission
electron microscopy, was not successful in discerning the structural
basis of the phenotype. The phenotype is associated with a
clumping of chloroplasts and other organelles around the nucleus.
It may involve some disruption of the cytoskeleton. The pleiotropic
effect of reduced spore viability is associated with somewhat fragile
spore walls. Different degrees or strengths of the clumping
phenotype are positively associated with increased spore wall
weakness.
Vaughn, K. C., L. G. Hickok, T. R. Warne, and A. C. Farrow. 1990.
Structural analysis and inheritance of a clumped-chloroplast mutant
in the fern Ceratopteris. Journal of Heredity 81:146–151.
Mutant: pale; gene symbols, pal1
Ghost-like pal1 gametophytes appear to have decreasing amounts
of chlorophyll as they enlarge. Nonetheless, they grow to sexual
maturity within 2 weeks and can self- or cross-fertilize. At 1–2
weeks, pal1 gametophytes are slightly smaller than wild type, and
their pale phenotype makes them clearly recognizable in a
segregating population (use of transmitted light is best). This is a
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Sport Reports - Descriptions of C-Fern Mutants
recessive mutation in sporophytes, and leaves of pal1/pal1
homozygotes show a yellow green phenotype when viewed with
reflected light (from the side or top). These attractive sporophytes
are quite viable, but slower growing than wild type. This EMSinduced mutation is available only in an F1 hybrid stock. The F
spores can be used very effectively as a substitute for polka dot
(cp) in basic Mendelian genetic labs. An additional pale mutant
(pal2) is available in combination with pal1. These mutants are
non-allelic and unlinked. Segregation from the F11results in a 1:3
(green:pale) gametophyte segregation ratio. Random fertilizations
produce an F2 sporophyte ratio of 9:7 (green:pale).
Mutant: dark germinator; gene symbol, dkg1
This is a fascinating mutation that shows a reversal in the light
requirement for germination of C-Fern spores. In wild type, light
(red) is necessary for spore germination. However, in spores
containing the dkg1 mutation, germination occurs readily in the
complete absence of light. Use of this mutant allows investigations
of gametophyte growth and development to be conducted in the
dark or by using specific wavelengths of light without the
requirement to expose spores to red or white light to initiate
germination. Another interesting aspect of this mutation is that
germination in white light is substantially reduced relative to its
germination in the dark.
Cooke, T., L. Hickok, W. J. Vanderwoude, J. Banks, and R. Scott.
1993. Photobiological characterization of a spore germination
mutant with reversed photoregulation in the fern Ceratopteris
richardii. Photochemical Photobiology 57:1032–1041.
Cooke, T. J., L. G. Hickok, and M. Sugai. 1995. The fern
Ceratopteris richardii as a lower plant model system for studying
the genetic regulation of plant photomorphogenesis. International
Journal of Plant Science 156:367–373.
Mutant: non-etiolated; gene symbol, det30
If C-Fern gametophytes are given an initial light exposure (1–2
days under standard conditions) to initiate germination and then
placed in the dark, their growth form will be dramatically altered.
The alteration in some ways resembles the etiolation response that
is well known for higher plants, in which plants subjected to dark
conditions grow substantially longer than those in light. In darkgrown C-Fern gametophytes, some of the basal cells undergo
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Sport Reports - Descriptions of C-Fern Mutants
extreme elongation, which results in strap-shaped structures
consisting of a few highly elongated cells just above the spore coat
and a pad of smaller cells at the tip of the gametophyte. In
gametophytes containing the det30 mutation, which was induced
by X-rays, this elongation response is reduced. In contrast to
flowering plants, both C-Fern wild type and det30 gametophytes
grown in the dark still have the capacity to synthesize chlorophyll,
although they are typically a very light green.
Cooke, T. J., R. H. Racusen, L. G. Hickok, and T. R. Warne. 1987.
The photocontrol of spore germination in the fern Ceratopteris
richardii. Plant and Cell Physiology 28:753–759.
Cooke, T. J., L. G. Hickok, and M. Sugai. 1995. The fern
Ceratopteris richardii as a lower plant model system for studying
the genetic regulation of plant photomorphogenesis. International
Journal of Plant Science 156:367–373.
Murata, T., A. Kadota, and M. Wada. 1997. Effects of blue light on
cell elongation and microtubule orientation in dark-grown
gametophytes of Ceratopteris richardii. Plant Cell Physiology
38:201–209.
Mutant: day-night responder; gene symbol, dnr1
Gametophytes and homozygous sporophytes containing this
mutation accumulate massive amounts of starch in their plastids
when grown under constant light conditions. This can be easily
observed by viewing the plastids under a compound microscope;
staining with I2KI can enhance observations. The massive starch
grains give the plastids a very lumpy appearance. The
accumulation of starch suggests that under constant light
conditions the cells are unable to use photosynthate effectively and,
as a result, gametophyte growth is severely impaired. This is a
conditional mutation in that near-normal growth and depletion of the
abnormal starch accumulation occur when gametophytes are
grown under day-night conditions. Sporophytes grow well under
normal greenhouse day-night conditions. This X-ray induced
mutation is recessive in sporophytes.
Mutant: abscisic acid tolerant; gene symbol, abr48
This mutation confers tolerance to the typical effects of abscisic
acid (ABA) on gametophytes. Wild-type gametophytes cultured in
the presence of ABA exhibit multiple effects involving decreased
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Sport Reports - Descriptions of C-Fern Mutants
growth rate and altered development. The number of antheridia on
hermaphroditic wild-type gametophytes is reduced and the number
of rhizoids is increased. In some wild-type hermaphrodites, the
presence of ABA can cause the marginal notch meristem to
become more centrally located. This results in an interesting
pattern of growth that resembles a tube sock, with the tip of the toe
representing the position of the meristem. The number of male
gametophytes is also reduced. These effects, which can be
induced in the wild type at a concentration of 5.0–50.0 mM ABA,
are not evident in the mutant.
Hickok, L. G. l985. Abscisic acid-resistant mutants in the fern
Ceratopteris: Characterization and genetic analysis. Canadian
Journal of Botany 63:1582–1585.
Warne, T. R., and L. G. Hickok. 1991. Control of sexual
development of Ceratopteris richardii: antheridiogen and abscisic
acid. Botanical Gazette 152:148–153.
Mutant: maleless; gene symbol, her1
The her1 mutation, which is one of many mutations that affect
sexual differentiation in C-Fern, was induced by X-irradiation of
spores. The effects of this mutation can be seen clearly in
populations of gametophytes. Populations of wild-type C-Fern
gametophytes show two sexual types, males and hermaphrodites.
In contrast, this maleless mutant does not contain male
gametophytes in populations. This mutation renders gametophytes
insensitive to the presence of the male-inducing pheromone,
antheridiogen (ACe). On the other hand, development of
hermaphrodite gametophytes occurs quite normally and appears no
different from the wild type. It is also possible to see the effects of
the mutation by using staggered sowings, over time, of spores on
the same petri dish. For direct testing, it is possible to obtain a
crude source of antheridiogen by growing multispore cultures of
gametophytes for 2–3 weeks on basic C-Fern medium and then
extracting the liquid from the medium. Extraction can be readily
done by taking the older cultures, scraping most of the
gametophyte material away, and then freezing the remaining agar.
After freezing, the agar can be thawed to effectively separate the
solid portion from the liquid. The solution can then be filtered and
used as a portion of the liquid to formulate new antheridiogensupplemented C-Fern medium, which can be referred to as
CFM+A. If spores of the wild type are sown on CFM+50% ACe, most
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Sport Reports - Descriptions of C-Fern Mutants
of the gametophytes in the culture will be male. In contrast, if
spores carrying the maleless her1 mutation are sown on such
medium, all of the gametophytes will be hermaphrodites.
Banks, J. A., L. Hickok, and M. A. Webb. 1993. The programming
of sexual phenotype in the homosporous fern, Ceratopteris
richardii. International Journal of Plant Science 154:522–534.
Banks, J. A. 1997. Sex determination in the fern Ceratopteris.
Trends in Plant Science 2:175–179.
Warne, T. R., L. G. Hickok, and R. J. Scott. 1988. Characterization
and genetic analysis of antheridiogen insensitive mutants in
Ceratopteris richardii. Botanical Journal of the Linnean Society
96:371–379.
Mutant: highly male; gene symbol, him1
Initial characterization of this EMS-induced mutation suggests that
it is highly sensitive to the pheromone, ACe. Typically, C-Fern
gametophytes always develop as hermaphrodites when cultured as
isolates. In contrast, him1 types can spontaneously develop as
males in isolate culture, and there are correspondingly higher
numbers of males in multispore cultures. In addition, him1
hermaphrodites tend to have much higher numbers of antheridia,
especially as they age. The higher numbers of males and
antheridia combine to produce cultures that have the capacity to
produce many sperm that can be observed in large masses.
Banks, J. A., L. Hickok, and M. A. Webb. 1993. The programming
of sexual phenotype in the homosporous fern, Ceratopteris
richardii. International Journal of Plant Science 154:522–534
Banks, J. A. 1997. Sex determination in the fern Ceratopteris.
Trends in Plant Science 2:175–179.
Warne, T. R., L. G. Hickok, and R. J. Scott. 1988. Characterization
and genetic analysis of antheridiogen insensitive mutants in
Ceratopteris richardii. Botanical Journal of the Linnean Society
96:371–379.
Mutant: salt tolerant; gene symbol, stl2
This mutation, which was induced with X-rays, shows an interesting
collection of co-segregating traits. It was selected for tolerance to
salt (NaCl), and gametophytes carrying the mutation show a high
level of tolerance to this agent. Sporophytes also show NaCl
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tolerance, but at a lower level and in a semi-dominant fashion. In
addition, stl2 confers tolerance to magnesium salts, but sensitivity
to moderate levels of potassium in the medium. Sensitivity of the
wild type to sodium salts is associated with the level of calcium in
the medium. In the presence of 150 mM NaCl, the calcium level in
basic C-Fern medium results in very poor growth and necrosis of
the wild type with little apparent effect on the mutant. At higher
calcium levels commonly found in other plant nutrient formulations,
the wild type and stl2 show similar responses at 150 mM NaCl. This
mutation can be used to show a simple salt tolerance response and
also can be used to demonstrate the complexities of mineral and
nutrient interactions in the environment.
Vogelien, D. L., L. G. Hickok, and T. R. Warne. 1996. Differential
effects of Na, Mg2+, K+, Ca2+and osmotic stress on the wild type and
NaCl-tolerant mutants, stl2, of Ceratopteris richardii. Plant, Cell and
Environment 19:17–23.
Warne, T. R., L. G. Hickok, T. B. Kinraide, and D. L. Vogelien.
1996. High salinity tolerance of the stl2 mutation of Ceratopteris
richardii is associated with enhanced K+ influx and loss. Plant, Cell
and Environment 19:24–32.
Mutant: FUDR tolerant; gene symbol, fdr1
This mutation, which was induced by X-rays, confers tolerance to
2'-deoxy-5fluorouridine (FUDR). This substance is a pyrimidine
nucleoside analog and is highly toxic to many organisms. As a
nucleotide analog, FUDR interferes with normal metabolism and
nucleic acid biosynthesis. This mutation confers a very clear
tolerance to FUDR and when gametophytes carrying it are grown in
medium containing 10 mM FUDR, wild-type gametophytes are
killed shortly after germinating while mutant gametophytes grow
quite normally.
Wu, K., and J. King. 1994. Biochemical and genetic
characterization of 5fluro-2'-deoxyuridine-resistant mutants of
Arabidopsis thaliana. Planta 194:117–122.
Mutant: sleepy sperm; gene symbol, zzz1
Wild-type C-Fern sperm, when released from the antheridium, are
encased in a thin-walled vesicle. Several seconds after release, the
sperm break free of the vesicle and quickly swim away. In contrast,
most zzz1 sperm remain in the vesicles after release and some
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eventually break free only after several minutes. The free sperm
typically move only slightly or swim very slowly to moderately, with
some showing faster movement. The more normal types apparently
allow zzz1 gametophytes to self fertilize, despite the abnormalities
in most zzz1 sperm. This EMS-generated mutant is excellent for
observations at both low and high (>50) magnifications.
Duckett, J. G., E. J. Klekowski, and L. G. Hickok. 1979.
Ultrastructural studies of mutant spermatozoids in ferns. I. The
mature nonmotile spermatozoid of mutation 230X in Ceratopteris
thalictroides (L.) Brongn. Gamete Research 2:317–343.
Mutant: slow-mo sperm; gene symbol, slo1
Like wild type, slo1 sperm are released from the antheridium in
thin-walled vesicles and then, shortly after release, break free and
swim away. However, most slo1 sperm swim very slowly and can
be easily observed, even under high (>50×) magnification. A few
moderately fast-swimming types allow for selffertilization of slo1
gametophytes. This mutation is EMS generated.
Duckett, J. G., E. J. Klekowski, and L. G. Hickok. 1979.
Ultrastructural studies of mutant spermatozoids in ferns. I. The
mature nonmotile spermatozoid of mutation 230X in Ceratopteris
thalictroides (L.) Brongn. Gamete Research 2:317–343.
Mutant: bubbles; gene symbol, bub1
This EMS-induced mutant appears normal until day 10, but then
begins to show massive swelling of a few to many cells in the
gametophyte. Some cells become so large that they actually burst.
Expression is variable but easy to observe. Self-fertile. Some
irregularity can also be seen in sporophytes that show variable
levels of penetrance and expressivity.
Mutant Hunt Mix
This mixture of wild type and a variety of mutant stocks (many
uncharacterized) is an excellent way to introduce students to the
wide variety of EMS-induced visible mutant types that can be
observed in C-Fern gametophytes. Mutants such as albino, pale,
polka dot, early gametophyte lethals, and a variety of other types
are present in a combined frequency of at least 10%. Some are
very easily observed, while others require more careful
observations. Using isolation and selfing or crossing techniques,
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students can discover, confirm, and characterize mutant
phenotypes and determine dominance/recessiveness.
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Glossary of Selected Terms
Glossary of Selected Terms
abscisic acid—a plant hormone; it reduces growth and sensitivity to
antheridiogen in C-Fern gametophytes.
allele—one of the alternative forms of a gene that occurs at the
same locus.
alternation of generations—life cycle alternating between
gametophytes and sporophyte.
antheridiogen—in ferns, a chemical substance (pheromone)
secreted by gametophytes that causes immature gametophytes to
develop as males.
antheridium (a)—a male sexual organ that produces sperm
(spermatozoids); the sperm-producing sex organ of seedless plants
as well as some fungi and algae.
archegonial neck—the nipple-shaped external portion of an
archegonium below the base of which the egg is located.
archegonium (a)—a female sexual organ that contains a single
egg; the egg-producing sex organ of seedless plants, such as ferns
and mosses.
buffer—substance that can stabilize the pH of a solution by
neutralizing the effects of acids or bases.
cell—the fundamental structural unit of living organisms.
cell wall—the exterior portion of the plant cell that surrounds the
protoplast.
chemoattractant—a chemical that can attract motile cells such as
sperm.
chlorophyll—the green pigment in plant cells used in
photosynthesis.
chloroplast—a chlorophyll-containing organelle (i.e., plastid) in
plants and algae.
circinate vernation—the specialized pattern of leaf development
that occurs in ferns, in which the leaf unrolls from the “fiddlehead.”
cultivar—a type of plant produced by artificial means through
cultivation, not existing naturally.
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Glossary of Selected Terms
clone—an exact copy of an individual.
cytoplasm—the protoplasm of a cell, not including the nucleus.
diploid—having two sets of chromosomes.
dominant—in genetics, a character state (or the allele that controls
it) that is expressed in the phenotype even if another allele for the
locus is present.
dormant—inactive, not growing.
egg cell—the female gamete.
embryo—the developing diploid sporophyte resulting from division
of the zygote.
etiolation—a growth response to the absence of light in which
certain cells become elongated; in higher
plants chlorophyll synthesis is also terminated.
fern—a seedless and flowerless plant that has two life-cycle stages
—a vascular sporophyte with roots, stems, and fronds (leaves),
which produces spores by meiosis and an independent, nonvascular, microscopic gametophyte stage that produces male and
female gametes.
fertilization—the union of nuclei from female and male gametes.
foot—the portion of the fern embryo that is embedded in the
gametophyte.
frond—a term used to denote the leaf in ferns.
gamete—a haploid reproductive or sex cell that can fuse with
another gamete to form a zygote.
gametophyte—the microscopic haploid phase of the fern life cycle
that produces gametes.
gene—a unit of heredity carried on the chromosome.
genotype—the genetic (allelic) makeup of an individual.
germination—the beginning of growth or development of a spore or
seed.
haploid—having one set of chromosomes.
hermaphrodite—a gametophyte containing a meristem and both
antheridia and archegonia.
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Glossary of Selected Terms
hermaphroditic—an organism with both male and female
reproductive organs.
heterosporous—producing two types of spores as a result of
meiotic division.
heterozygous—having two different alleles at the same locus.
homosporous—producing one type of spore as a result of meiotic
division.
homozygous—having two identical alleles at the same locus.
hybrid—a combination of two different types.
imbibition—absorption of fluid by physical means.
incompatibility—in plants, a situation in which a particular genetic
combination is not viable.
leaf—the major photosynthetic organ of vascular plants.
locus—the location of a gene on a chromosome.
male—a gametophyte lacking a meristem and containing numerous
antheridia.
meiosis—a process of cell division in sexually reproducing
organisms that results in reduction of chromosome number (by 2) in
the products.
meristem—an area of active cell division that gives rise to other
cells and tissues. mineral nutrient—an inorganic substance
necessary for normal growth.
mutant—a genetic variant.
neck canal cells—a row of cells present in the neck of an unopened
archegonium.
pH—a measurement of the acidity or relative concentration of
hydrogen ions in a solution (pH=-log[H+]). A neutral solution is pH
7, an acidic solution less than 7, and an alkaline or basic solution
greater than 7.
phenotype—the appearance of an organism, which results from the
interaction of its genotype and the environment.
pheromone—a substance produced by one individual of a species
that affects the development of other individuals in a population.
phloem—the food-conducting tissue of a vascular plant.
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Glossary of Selected Terms
photosynthesis—a biochemical process in green plants by which
(typically) carbohydrates are synthesized from carbon dioxide and
water using light as an energy source and releasing oxygen as a
by-product.
pigment—any of a number of organic molecules that absorb and
reflect light of particular wavelengths.
polyploid—having more than double the basic chromosome
number.
prothallus (also prothallium)—a structure produced from a
developing spore that bears sex organs;sometimes used
interchangeably with “gametophyte.”
pteridophyte—a term commonly referring to ferns and fern allies
such as horsetails and club mosses.
qualitative data—data expressed as a description of an inherent or
distinguishing characteristic, property, or trait.
quantitative data—data expressed as number, measurement, or
quantity.
recessive—in genetics, a character state (or the allele that conrols
it) that is expressed in the phenotype only if another identical allele
for the locus is present. A recessive allele can be masked by a
dominant allele.
rhizoid—a clear, thread-like cell visible in germinating spores and at
the base of gametophytes.
root—the vascular organ of sporophytes that is typically associated
with the substrate.
root cap—the very tip portion of a root that is composed of a loose
aggregation of friable cells.
sample (sampling)—in statistics, a subset which is analyzed to be
representative of the whole population. seed plant—a plant that
bears seeds.
segregation—in genetics, the separation during meiosis of a pair of
alleles at a given locus, resulting ing ametes containing only a
single allele per locus.
sexual reproduction—reproduction based on the reciprocal
processes of meiosis and fertilization.
shoot—the arial portion of a plant distinct from the root.
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Glossary of Selected Terms
species—a group of interbreeding individuals with similar
characteristics.
sperm (spermatozoid)—in ferns, a motile male gamete.
spore—single-celled haploid (1n) product of meiosis that
germinates and develops into a gametophyte.
spore mother cell—a diploid (2n) cell that undergoes meiosis to
produce haploid (1n) spores.
sporophyte—the macroscopic diploid vascular stage of the fern life
cycle that produces spores.
strain—a group of plants distinguished by a particular trait.
syngamy—the fusion of two gametes. variation—a deviation from
the norm or standard type.
wet mount—a preparation of tissues or cells placed in water on a
slide and covered with a coverslip to allow viewing under a
compound microscope.
wild type—the phenotype selected as a standard for comparison
with other phenotypes; often the predominant phenotype among
individuals of species.
xylem—the water-conducting tissue of a vascular plant.
zygote—the diploid cell produced by the fusion of gametes.
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