Undergraduate Thesis Defense Slides
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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!!!