Density-Dependent Sexual Differentiation in Single-Nuclear

Mutants of Ceratopteris

richardii

Density-Dependent Sexual Differentiation in Single-Nuclear

Mutants of Ceratopteris

richardii

Heather E. Jordan

Biology Department

College of Health and Natural Sciences

Lake Superior State University

Sault Ste. Marie, MI 49783

204/11/23

IntroductionIntroduction

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What is Ceratopteris richardii?What is Ceratopteris richardii?

- A semi-aquatic, homosporous fern

- Commercially available in a variety of mutant stocks

- Sex ratios of the gametophytes are subject to population pressures

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Why study C. richardii?Why study C. richardii?

Wide variety of mutant strains

Large sample sizes require little space

Grow quickly Swimming

sperm makes it interesting to “anti-botanists”

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Notes of Interest:Notes of Interest: As the growth density increases, more ACe is secreted

into the surrounding medium. Ace causes developing gametophytes to become male Therefore, growth density leads to in the

percentage of males in a population.

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Factors Influencing the Effects of the Pheromone on the Sex Ratio:

Factors Influencing the Effects of the Pheromone on the Sex Ratio:

Growth rate of gametophytes

Production and diffusion of the antheridiogen

Influence of growth promoters and inhibitors

Gametophytic growth density

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Project ObjectiveProject Objective Test the effects of various types of single gene

nuclear mutations on density-dependent sexual differentiation in C. richardii.

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HypothesisHypothesis Populations of mutant strains not bred to exhibit an

altered response to the antheridiogen ACe will yield sex ratios similar to those found in a wild type population.

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Summary of Strains InvestigatedSummary of Strains Investigated

GenotypeCode Generic NameRNWT 1 Wild Typeabr48 Abscisic Acid Tolerantcp Polka Dotdet30 Non-Etiolatedfdr1 FUDR Toleranther1 Malelesshim1 Highly Malerum1 Rumpledslo1 Slo-Mo Spermstl2 Salt Tolerantzzz1 Sleepy Sperm

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Methods & Materials

Methods & Materials

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Sowing The Spores:Sowing The Spores: To synchronize germination,

the spores were soaked in 4 mL of distilled water for a week in complete darkness.

Since 1 drop contains about 100 spores, different growth densities can be obtained by adjusting the number of drops used to inoculate each plate.

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Experimental setup of the culture dome for each genotype:

Experimental setup of the culture dome for each genotype:

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Temperature: 28oC pH = 6 Photoperiod: 24 hourTemperature: 28oC pH = 6 Photoperiod: 24 hour

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How do you know which ones are male?How do you know which ones are male?

Male Cordate

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Counting MethodsCounting Methods1. A quadrant was

selected at random.

2. All gametophytes were counted and sexed in that quadrant.

3. Obtained numbers were multiplied by 4 for a final estimate.

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To make the counting a little easier…To make the counting a little easier…

Projection onto a computer monitor using the Intel QX3 Microscope.

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Statistical AnalysisStatistical Analysis

Single Factor Model I ANOVA’s between the 11 strains were calculated for:

1. The mean number of spores germinated

2. The mean percentage of males present.

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ResultsResults

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Could there be a trend?Could there be a trend?Strain Mean # of

GametophytesMean % Male

RNWT1 360.9 34.6

rum1 281. 2 19.8

fdr1 255.2 28.9

abr48 249.3 36.5

stl2 249.3 15.4

her1 211.9 0.00

slo1 157.1 13.9

him1 140.8 98.8

det30 136.3 13.5

zzz1 135.9 17.6

cp 89.9 14.5

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F ValuesF Values

Fcrit Mean # Gametophytes Mean % Male

RNWT1 vs. rum1 4.01 1.66 11.39

RNWT1 vs. fdr1 4.01 2.80 1.51

RNWT1 vs. abr48 4.01 3.18 0.16

RNWT1 vs. stl2 4.01 2.92 18.30

RNWT1 vs. her1 4.01 6.06 87.16

RNWT1 vs. slo1 4.01 13.54 24.69

RNWT1 vs. him1 4.01 16.38 295.66

RNWT1 vs. det30 4.01 16.66 21.10

RNWT1 vs. zzz1 4.01 15.77 13.26

RNWT1 vs. cp 4.01 26.90 22.12

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The percentage of males increases with a rise in the population density across each strain.

The percentage of males increases with a rise in the population density across each strain.

Sexual Differentiation With in Each Strain

0

10

20

30

40

50

60

0 1000 2000 3000 4000 5000 6000 7000

Numb er of G am etop hy tes

% M

ale

RNWT1

abr48

stl2

rum1

zzz1

det30

cp

fdr1

s lo1

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The overall composition of each genotype sample:The overall composition of each genotype sample:

Population Composition

02000400060008000

10000

RNW

T1 rum

1

fdr1

abr4

8 stl2

her1

slo1

him

1

det3

0

zzz1 cp

Strain

# of

Gam

etop

hyte

s

Number ofMalesNumber ofCordates

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DiscussionDiscussion

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In Summary…In Summary… Differences observed in the percentage of males Differences observed in the percentage of males

found within each strain appears to be found within each strain appears to be dependent on dependent on how well a particular strain growshow well a particular strain grows under standard under standard conditons.conditons.

There were no significant differences between the There were no significant differences between the growth ratesgrowth rates of of chemically tolerantchemically tolerant mutants and the mutants and the wild type.wild type.

Significant differences in Significant differences in growth ratesgrowth rates were found in were found in mutants carrying genes for mutants carrying genes for structural mutationsstructural mutations..

Only Only 22 strains showed strains showed no significant differences in no significant differences in growth rategrowth rate but exhibited but exhibited significant differences in the significant differences in the percentage of malespercentage of males present in each sample (stl2 & present in each sample (stl2 & rum1).rum1).

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Chemical Tolerance (abr48, fdr1, and stl2)Chemical Tolerance (abr48, fdr1, and stl2)

Previous studies show significant differences in growth rates.

Typically, mutants grown in the absence of the chemical they were bred to resist do not grow as well as the wild type.

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Structural Mutations (det30, cp, slo1, and zzz1)

Structural Mutations (det30, cp, slo1, and zzz1)

These strains had the lowest germination rate.

The nature of the mutation may result in functional impairments.

Possibly a consequence of the mutagenic process itself.– Chemical treatment and/or radiation

of plant tissues has been shown to induce chromosome stickiness).

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Strains with altered differentiation patterns and normal growth rates

Strains with altered differentiation patterns and normal growth rates

This experiment did not test for:– antheridiogen production by the gametophytes– The influence of mutations on growth promoters

and inhibitors in the strains studied.

Perhaps testing the stl2 and rum1 strains for these things may be helpful in finding out if these two strains possess an altered sensitivity to the antheridiogen.

Hint, hint– Junior Seminar Students!!!