Transcript Gough - York College of Pennsylvania

Selected Stages of Megasporogenesis and Megagametogenesis in
Arabidopsis thaliana (L.) Heynh Colombia Ecotype
Les Gough
Introduction
Arabidopsis thaliana (L.) Heynh Colombia Ecotype is a
small flowering plant of the mustard (Brassicaceae) family. A.
thaliana ecotypes have been maintained and described from
around the world. A. thaliana is best known a laboratory
organism and is widely used as a model organism in plant
biology. This is the first quantitative embryological study of A.
thaliana (L.) Heynh Colombia Ecotype. I quantified three stages
of megasporogenesis and three stages of megagametogenesis.
The three stages of megasporogenesis are the megaspore mother
cell (MMC) that produces a dyad after meiosis I. As the second
meiotic division progresses, a tetrad is formed. The chalazal
spore enlarges to become the embryo sac and ends
megasporogenesis (Smith 1981). The functional megaspore, after
the first mitotic divisions, becomes a 2-nucleated embryo sac and
a second division produces a 4-nucleated embryo sac.
Megagametogenesis ends when the third mitotic division
produces an 8-nucleated embryo sac and is ready for fertilization
of the egg and the polar nuclei.
Department of Biological Sciences, York College of Pennsylvania
•Seeds were planted using growing instructions from the Wisconsin
Fast Plants Manual (Carolina Biological Supply Company catalog,
2003-2004).
•2 weeks after planting, the earliest floral buds were collected and
fixed in FPA50 (Formalin, propionic acid, and 50% ethyl alcohol
[ETOH]).
4-Nucleate
Dyad
•Micropipettes were used to move dissected ovules to a Raj slide.
2-Nucleate
MMC
•All dissections and placements of ovules on Raj slides are done using a Nikon SMZ1000 Zoom Stereo Microscope and a Nikon E600 Phase Contrast Microscope.
FM
0
5
10
15
20
25
0
10
Length (um)
•Images were captured using a Nikon DXM1200F digital camera, and analyzed using
Nikon ECLIPSENET software.
Fig. 10
Megasporogenesis
Dyad
2-Nucleate
MMC
FM
Fig.7
8
12
Width (um)
30
40
Fig. 11 Megagametogenesis
4-Nucleate
4
20
Length (um)
Tetrad
0
16
20
0
4
8
12
16
20
Width (um)
Figures 8-11. Ten ovules were measured for each stage. The boxes represent the means
and the whiskers show the range.
Figure 7. Example of how length and width measurements were made. Lines were
measured and calibrated using ECLIPSENET.
Megasporogenesis
Fig.1
Fig.2
Length and width: The values for the minimum size, the mean, and
confidence intervals increase from the MMC through the Tetrad and decreases
only between the tetrad and the FM. Only the length’s standard deviation
decreases from the dyad to tetrad stage (Tables 1 and 2).
Fig.3
Megagametogenesis
Fig.4
Lengths and widths: The values for the minimum size, the mean, and
confidence intervals is smaller in all cases for the FM compared to the previous
tetrad stage, but increases for each measured stage beyond this point. The
standard deviations, for all stages, continue to increase (Tables 1 and 2).
Fig.6
Fig.5
Legend
Fig.1. Megaspore mother cell (MMC) stage, Fig.2. Dyad stage of
meiosis, Fig.3. Tetrad stage of meiosis, Fig.4. Functional megaspore
(FM)-chalazal, Fig.5. Two Nucleate stage, and Fig.6. Four nucleate
stage
Results
Discussion
Table 1. Smallest and largest measurements, mean, standard deviation and 95% confidence intervals for
length.
Min-Max
MMC
Dyad
Tetrad
FM
2-Nuc
4-Nuc
10.80-13.92
12.74-16.02
14.66-17.43
12.68-18.79
18.82-24.39
24.25-37.56
Mean
12.80
14.02
16.31
15.73
21.70
32.44
SD
0.8850
0.9666
0.8572
1.806
1.961
3.955
•Whole flowers were placed in a dehydration series of 80%, 90%,
95%, to 100% ETOH
CI
12.16-13.43
13.33-14.72
15.70-16.93
14.43-17.02
20.30-23.11
29.61-35.27
•The ovary remained in each alcohol concentration for a minimum of
10 min and were placed in the Herr Clearing Fluid (HCF=chloral
hydrate, phenol, lactic acid, clove oil, and xylene, 2:2:2:2:1 by
weight).
Table 2. Smallest and largest measurements, mean, standard deviation and 95% confidence intervals for
width.
•Ovaries remained in HCF for a minimum of 24 h before viewing.
•Whole ovaries were placed in a glass planchet and immersed with
HCF for dissection of ovules.
Min-Max
•Collections continued for six weeks and fixations lasted for a
minimum of 24 h followed by permanent storage in 70% ETOH.
Fig. 9 Megagametogenesis
Tetrad
Methods (Continued)
Much of the success of this experiment and other previous
experiments is credited to the development of 4 1/2 clearing fluid
(Herr clearing fluid). New morphological data may provide
insight into the range of variation expressed by this genus and a
more precise concept of species relationships (Smith and Herr
1971). This study was undertaken to: (a) find and measure the
lengths and widths of at least ten examples of the three stages of
megasporogenesis and the three stages of megagametogenesis in
A. thaliana (L.) Heynh Colombia Ecotype; (b) compute means
and confidence intervals of those measurements.
Methods
Megasporogenesis
Fig. 8
MMC
Dyad
Tetrad
FM
2-Nuc
4-Nuc
7.470-9.740
8.610-10.89
9.940-12.40
7.050-11.69
9.970-13.89
13.01-17.85
Mean
8.855
9.666
11.12
9.210
11.65
15.66
SD
0.7842
0.6701
0.7660
1.389
1.282
1.449
9.187-10.15
10.57-11.67
8.216-10.20
10.73-12.56
14.62-16.70
CI
8.294-9.416
Comparing these measurements indicate the variation in growth between
the successive stages. The difference in means between successive stages
indicates the amount of growth between stages (Smith 1971). The means of
length and width increased with advancing stages of development except for
the functional megaspore. This growth pattern is expected because during
megasporogenesis, meiosis 1 & 2 occur producing a tetrad. The decrease in
mean growth at the FM is a result of the crushing of the remaining tetrad.
Growth begins to increase, as megagametogenesis goes through mitosis 1 & 2.
Overlapping of 95% confidence intervals between successive stages indicate
uniform growth between stages of development. Future studies, of this type,
should compare measurements among the many ecotypes of A. thaliana.
Literature Cited
Smith, B.B. Comparisons of Early Ovule Development through Megagametogenesin
Rapid-cycling Brassica rapa L. and B. campestris L. Using the Herr Clearing
Techniques. In: E. Otaviano et al. Eds. Angiosperm Pollen and Ovules. SpringerVerlag. 1992
Smith, B.B. and Herr, J.M. Jr. 1971. Ovule Development, Megagametogenesis, and
Embryogeny in Ammania Coccinea Rothb. The Journal of the Elisha Mitchell
Scientific Society. 87(4):192-199.
Acknowledgements: I would like to thank Dr. Smith for his help and guidance throughout my research.