Selected Stages of Megasporogenesis and Megagametogenesis in Arabidopsis thaliana (L.) Heynh...
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Selected Stages of Megasporogenesis and Megagametogenesis in Arabidopsis thaliana (L.) Heynh Colombia Ecotype Les Gough Department of Biological Sciences, York College of Pennsylvania 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. 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 •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 FPA 50 (Formalin, propionic acid, and 50% ethyl alcohol [ETOH]). •Collections continued for six weeks and fixations lasted for a minimum of 24 h followed by permanent storage in 70% ETOH. •Whole flowers were placed in a dehydration series of 80%, 90%, 95%, to 100% ETOH •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). •Ovaries remained in HCF for a minimum of 24 h before viewing. •Whole ovaries were placed in a glass planchet Resul ts Discussion 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. Table 1. Smallest and largest measurements, mean, standard deviation and 95% confidence intervals for length. MMC Dyad Tetrad FM 2-Nuc 4-Nuc Min-Max 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 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 Table 2. Smallest and largest measurements, mean, standard deviation and 95% confidence intervals for width. MMC Dyad Tetrad FM 2-Nuc 4-Nuc Min-Max 7.470-9.740 8.610-10.89 9.940-12.40 7.050- 11.69 9.970-13.89 13.01-17.85 Acknowledgements: I would like to thank Dr. Smith for his help and guidance throughout my research. 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. Springer- Verlag. 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. Methods (Continued) •Micropipettes were used to move dissected ovules to a Raj slide. •All dissections and placements of ovules on Raj slides are done using a Nikon SMZ-1000 Zoom Stereo Microscope and a Nikon E600 Phase Contrast Microscope. •Images were captured using a Nikon DXM1200F digital camera, and analyzed using Nikon ECLIPSENET software. Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 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 Megasporogenesis 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). Megagametogenesis 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). Figure 7. Example of how length and width measurements were made. Lines were measured and calibrated using ECLIPSENET. Fig.7 Figures 8-11. Ten ovules were measured for each stage. The boxes represent the means and the whiskers show the range. M egasporogenesis 0 5 10 15 20 25 MMC Dyad Tetrad Fig. 8 Length (um ) M egagam etogenesis 0 10 20 30 40 FM 2-Nucleate 4-Nucleate Fig. 9 Length (um ) M egasporogenesis 0 4 8 12 16 20 MMC Dyad Tetrad Fig. 10 W idth (um ) M egagam etogenesis 0 4 8 12 16 20 FM 2-Nucleate 4-Nucleate Fig. 11 W idth (um )
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Transcript of Selected Stages of Megasporogenesis and Megagametogenesis in Arabidopsis thaliana (L.) Heynh...
Selected Stages of Megasporogenesis and Megagametogenesis in Arabidopsis thaliana (L.) Heynh Colombia Ecotype
Les Gough
Department of Biological Sciences, York College of PennsylvaniaIntroduction
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.
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
•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]).
•Collections continued for six weeks and fixations lasted for a minimum of 24 h followed by permanent storage in 70% ETOH.
•Whole flowers were placed in a dehydration series of 80%, 90%, 95%, to 100% ETOH
•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).
•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.
Results Discussion
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.
Table 1. Smallest and largest measurements, mean, standard deviation and 95% confidence intervals for length.
MMC Dyad Tetrad FM 2-Nuc 4-Nuc
Min-Max 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
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
Table 2. Smallest and largest measurements, mean, standard deviation and 95% confidence intervals for width.
MMC Dyad Tetrad FM 2-Nuc 4-Nuc
Min-Max 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
CI 8.294-9.416 9.187-10.15 10.57-11.67 8.216-10.20 10.73-12.56 14.62-16.70 Acknowledgements: I would like to thank Dr. Smith for his help and guidance throughout my research.
Literature CitedSmith, 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. Springer- Verlag. 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.
Methods (Continued)
•Micropipettes were used to move dissected ovules to a Raj slide.
•All dissections and placements of ovules on Raj slides are done using a Nikon SMZ-1000 Zoom Stereo Microscope and a Nikon E600 Phase Contrast Microscope.
•Images were captured using a Nikon DXM1200F digital camera, and analyzed using Nikon ECLIPSENET software.
Fig.1 Fig.2 Fig.3
Fig.4 Fig.5 Fig.6
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
Megasporogenesis
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).
Megagametogenesis
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).
Figure 7. Example of how length and width measurements were made. Lines were measured and calibrated using ECLIPSENET.
Fig.7
Figures 8-11. Ten ovules were measured for each stage. The boxes represent the means and the whiskers show the range.
Megasporogenesis
0 5 10 15 20 25
MMC
Dyad
Tetrad
Fig. 8
Length (um)
Megagametogenesis
0 10 20 30 40
FM
2-Nucleate
4-Nucleate
Fig. 9
Length (um)
Megasporogenesis
0 4 8 12 16 20
MMC
Dyad
Tetrad
Fig. 10
Width (um)
Megagametogenesis
0 4 8 12 16 20
FM
2-Nucleate
4-Nucleate
Fig. 11
Width (um)
