A Quantitative Analysis of Megagametogenesis in
Two Species of Arabidopsis thaliana.
Kasey Sanders
Department of Biological Sciences, York College of Pennsylvania
Figure 1. Arabidopsis thaliana after flowering.
http://commons.wikimedia.org/wiki/File:Arabidopsis_thaliana_i
nflorescencias.jpg
Introduction
Methods
• Arabidopsis thaliana, a flowering plant found in the mustard
(Brassicaceae) family (Figure 1), is a widely used biological model
due to its rapid life cycle and abundant seed production. It was
the first plant to have its genome mapped and offers important
advantages in genetic and molecular biology (ABRC, 2012).
Seeds were ordered,
planted in individual
wells, and each well was
assigned a number.
Figure 3. Map of Japan. Cities of study include Sapporro (Northern) and Kyoto City
(Southern), Japan and are marked by large red spots on map.
http://www.lonelyplanet.com/maps/asia/japan/
Figure 2. Stages of Megagametogenesis. This includes the
functional megaspore, 2-Nucleate, 4-Nucleate, and 8-Nucleate.
http://agritech.tnau.ac.in/seed/seedconcepts.html
• Ecotypes of Arabidopsis thaliana have shown variations in
flowering in response to growing conditions. These variations
affect development of the haploid generation which can be seen
by conducting microscopic studies of this generation (Smith,
1973).
Results
Dehydrated in
series of EtOH
(70, 80, 85, 90, 95,
100%)
Flowers were collected
and fixed in FPA50.
• Megagametogenesis is the development of the megaspore into
the embryonic sac which occurs in four stages: the functional
megaspore (FM), 2-Nucleate (2N), 4-Nucleate (4N), and 8nucleate (8N) (Figure 2).
Stored in Herr Clearing
Fluid for 24 hours.
• Two Japan ecotypes were studied; one from Kyoto City
(Northern) and one from Sapporo (Southern). Ecotypes from
these two cities were chosen because they differ in climate,
elevation, and growing conditions (Figure 3). Observations were
made to see if these differences cause changes in the
development of the haploid generation.
• The Fibonacci series is used to determine if the nucleus sizes of
the ecotypes of study correspond with expected results for
Arabidopsis thaliana by evaluating the mean length and width of
the nucleus. For the ratio to be met the mean width must be 1050.5% of the mean length of the nucleus (Herr, 1967).
• Phi (1.6180339) is referred to as the “golden ratio” because it is
the ratio by which many things develop. This ratio was proven to
be a defining characteristic of length and width of gametophyte
stages (Mitchison, 1977).
• For many ecotypes the three-decimal Phi is encountered at the
11th division and the seven-decimal Phi at the 18th division.
Encountering the “golden ratio” at a different division shows a
difference in development (Herr, 1967).
Objective
• Successfully grow two ecotypes of Arabidopsis
thaliana from Japan; one from Kyoto City
(Northern) and one from Sapporro (Southern).
• Determine if differences are present between
ecotypes by measuring mean length and width for
stages of Megagametogenesis.
• Using the Fibonacci Sequence the ecotypes will
compared to the golden ratio to observe
differences.
Hypothesis
H: The four stages of megagametogenesis will meet
the 3-decimal Phi at the 11th division and the 7decimal Phi at the 18th division and there will be no
difference between plants of the same ecotype.
H0: There will be a difference between the stages of
megagametogenesis for the two ecotypes and the
golden ratio Phi.
Ovules dissected from
pistil , stage
determined, and
measurements taken
using phase contrast
microscope.
Data was calculated
using a Fibonacci
sequence.
B
C
D
Figure 4. Images taken of the four stages of Megagametogenesis.
Circles indicate location of nucleus. A) Functional Megaspore
B) 2-Nucleate C) 4-Nucleate D)8-Nucleate.
Table 2. Fibonacci sequence for Plant 9
from Sapporo ecotype.
Table 1. Fibonacci Sequence for Plant 4 from
Kyoto City ecotype.
Ratio
A
Ratio
Division
Width:
15.59
Length:
48.65
3.120590122
1
64.24
1.320452210
2
112.89
1.757316314
3
177.13
1.569049517
4
290.02
1.637328516
5
467.15
1.610750983
6
757.17
1.620828428
7
1224.32
1.616968448
8
1981.49
1.618441257
9
3205.81
1.617878465
10
5187.30
1.618093399
11
8393.11
1.618011297
12
13580.41
1.618042656
13
21973.52
1.618030678
14
35553.93
1.618035253
15
57527.45
1.618033506
16
93081.38
1.618034173
17
150608.83
1.618033918
18
243690.21
1.618034016
19
394299.04
1.618033978
20
637989.25
1.618033993
21
1032288.29
1.618033987
22
1670277.54
1.618033989
23
2702565.83
1.618033989
24
4372843.37
1.618033989
25
Width
Length
13.74
40.71
54.45
95.16
149.61
244.77
394.38
639.15
1033.53
1672.68
2706.21
4378.89
7085.10
11463.99
18549.09
30013.08
48562.17
78575.25
127137.42
205712.67
332850.09
538562.76
871412.85
1409975.61
2281388.46
3691364.07
2.962882096
1.337509211
1.747658402
1.572194199
1.636053740
1.611226866
1.620645063
1.617038254
1.618414560
1.617888658
1.618089505
1.618012784
1.618042088
1.618030895
1.618035170
1.618033537
1.618034161
1.618033923
1.618034014
1.618033979
1.618033992
1.618033987
1.618033989
1.618033989
1.618033989
Division
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Conclusion
• The 3-decimal Phi was reached at the 11th division
and the 7-decimal Phi was reached at the 18th
division.
• This is what was expected and shows that the change
in environments does not have a large enough effect
on the haploid generation to cause differences in
development.
Literature Cited
•
•
•
•
•
ABRC. 2012. The Ohio State University. http://abrc.osu.edu/
Herr, J. 1967. “On the Nature of Variation.”
Phytomorphology, 17:200-207.
Mitchison, G.J. 1977. “Phyllotaxis and the Fibonacci Series.”
Science 196 (4287): 270-275.
Smith, Bruce B. 1973. "The Use of a New Clearing
Technique for the Study of Early Ovule Development,
Megasporogenesis, and Megagametogenesis in Five Species
of Cornus L." Amer.ican Journal of Botany 60(4): 322-38.
Smith, B, B. 1975. “A Quantitative Analysis of the
Megagametophyte of Five Species of Cornus L.” American
Journal of Botany 62(4): 387-394.
Acknowledgements
I would like to thank Dr. Smith for his guidance and expertise
throughout my project as well as my peers who assisted me
along the way.