Measurement of Selected Stages of Megasporogenesis, and Megagametogenesis in Arabidopsis thaliana Landsberg Erecta Ecotype Stephany McDonough Department of Biological Sciences, York College of Pennsylvania Arabidopsis thaliana is a small herbaceous annual of the Brassicaceae family. Several features of A. thaliana make it a widely used model organism in plant biology, and I chose to work with the Landsberg erecta ecotype. Embryology includes the successive stages of megasporogenesis (the meiotic development of the functional megaspore), megagametogenesis (the development of the female and male gametophyte), and the post-fertilization stages from the fertilized egg to the mature embryo (Bowman 1994). I quantified four stages of megasporogenesis and three stages of megagametogenesis. The four stages of megasporogenesis are the megaspore mother cell that yields, after meiosis I, a dyad and, after meiosis II, yields a tetrad. The chalazal member of the linear tetrad becomes the functional megaspore and ends megasporogenesis. The functional megaspore, after three mitotic divisions, becomes an 8-nucleated embryo-sac thus ready for fertilization of the egg and the polar nuclei and the end of megagametogenesis (Smith 1975). The development of a clearing technique developed by Dr. J.M. Herr (1971) made a huge impact on embryological studies. When the flowers were placed in the clearing fluid the tissue was cleared for easier microscopic viewing. New morphological data may provide insight into the range of variation expressed by this genus and a more precise concept of ecotype relationships (Smith and Herr 1971). The objectives of my research were to (a) Find and measure at least ten examples of the stages of megasporogenesis and megagametogenesis. (b) Once measured, means and confidence intervals will be computed for future comparative reference to other ecotypes. Introduction Abstract Seeds were planted. Approximately 2 weeks after planting, floral buds were collected and fixed in FPA 50 (Formalin, Propionic acid, Ethyl Alcohol-50%) for a minimum of 24 hours. Weekly collections were fixed for 6 weeks and each collection stored in 70% ETOH. When ready for clearing, whole flowers were dehydrated through an alcohol series % ETOH and… After 10 min in each solution the floral buds were placed in Herr Fluid. - Equal parts chloral hydrate, clove oil, phenol crystals, and lactic acid - Half the other ingredients xylene (2:2:2:2:1) After the flowers were in Herr fluid for 3-5 days they were ready for dissection. The Nikon stereo zoom dissecting microscope was used to remove all parts of the flower. Ovules were removed from the ovary. Each ovule was examined under the oil immersion lense of the Nikon E600 phase contrast microscope to find the development stage. All images were captured using the Nikon DXM1200 digital camera. Image J software was utilized for measurement purposes which included the max. length and width of each development stage (see fig.8) Data was collected for all 10 samples of each development stage and analyzed using Prisms software. Materials and Methods Figure 2. The dyad stage Figure 5. The 2-nucleate stage Figure 4. The functional megaspore (FM) stage Figure 1. Megaspore mother cell (MMC) stage Figure 3. The tetrad stage Figure 6. The 4-nucleate stage Figure 7. The 8-nucleate stage Figure 8. Length and width measurements. Lines were calibrated using Image J software. Figures Ten ovules were measured for each stage. The boxes represent the means and the wiskers show the range. The measurements made of the stages from A. thaliana indicate the variation in growth. A comparison of the size variation of the megagametophyte stages indicates that the greatest growth increment occurs during the 4- nucleate stage. The maximal length is probably in the 8- nucleate stage, but I was not able to distinguish this stage due to starch build up (fig.7). In megasporogenesis the greatest growth increment occurs during the dyad stage. The smallest size of each megagametophyte stage is less than the greatest size of the preceding stage in both length and width measurements. The mean measurements in Tables 1 and 2 show an increase through megasporogenesis, and then a decrease at the functional megaspore stage. Table 1. Smallest and largest measurements as well as the mean and standard deviation for lengths. MMC Dyad Tetrad FM 2-Nuc 4-Nuc Min-Max Mean SD Table 2. Smallest and largest measurements as well as the mean and standard deviation for widths. MMC Dyad Tetrad FM 2-Nuc 4-Nuc Min-Max Mean SD Results The data collected for A. thaliana clearly indicates that the size ranges of megagametophyte stages overlap. This overlap significantly indicates the nuclear divisions which establish the successive stages are not directly correlated with a specific growth increment of each stage (Smith and Herr 1971). The difference in means between successive stages indicates the amount of growth between stages. The decrease between successive means was between the tetrad and the functional megaspore stages. The most likely explanation for this is that the cells in the tetrad stage are squished and therefore the one FM cell is decreased in width and length. Discussion Another ecotype may be studied and compared to the Landsberg erecta. More samples may be used for more accurate results. Develop a clearing technique that eliminates starch, so that the 8-nucleate stage can be studied. Future Research Figure 9. Figure 10. Figure 11. Figure 12. Quantitative comparisons of variations of morphological features during megasporogenesis and megagametogenesis suggest differences in growth patterns. The objectives of my research were to find and measure ten examples of the three stages of megasporogenesis and the four stages of megagametogenesis. Once measured, means and confidence intervals were computed for future comparative reference to other ecotypes. Length and width measurements were graphed and showed increasing sizes through the succeeding stages. The differences in means between successive stages indicate the amount of growth between stages. Bowman, J. Editor Arabidopsis, An Atlas of Morphhology and Development. Springer-Verlag. New York. (AAMD) Smith, B.B A Quantitative Analysis of the Megagametophyte of Five Species of Cornus L. American Journal of Botany. 62(4): Smith, B.B. and Herr, J.M. Jr Ovule Development, Megagmetogenesis, and Embryogeny in Ammania Coccinea Rothb. The Journal of the Elisha Mitchell Scientific Society. 87(4): Starch granules Literature Cited Acknowledgements I would like to thank Dr. Smith for his help and guidance throughout my research.