The Reinvestigation of Ovule Development Through Megagametogenesis of the Rapid Cycling Brassica rapa L. and the Quantification of Various Stages William Hauck Department of Biological Sciences, York College of Pennsylvania ABSTRACT: Rapid cycling Brassica rapa L. was re-examined to qualitatively validate the stages of megasporogenesis and megagametogenesis reported in a previous study. Whole flowers were fixed in FPA 50 for a minimum of 24 h and stored in 70% ethyl alcohol. From storage, flowers were dehydrated to 100% ethyl alcohol. From 100% ethyl alcohol flowers were placed in Herr clearing fluid for a minimum of 24 hours. Ovules were dissected from the ovaries and placed on Raj slides and viewed with dissecting and phase contrast microscopy. Besides the qualitative validation of ovule stages, a quantitative study of the length and width of ovules and stages within those ovules were compared to the ovary length and width from which they were dissected. No correlation was present using Pearson’s Coefficient, which is possibly due to a small sample size yet trends existed when looking at the means of each stage area and its corresponding ovary and ovule areas. INTRODUCTION: Rapid cycling Brassica rapa was developed to facilitate the research and teaching of many different disciplines within the biology field by the Crucifer Genetics Cooperative at the University of Wisconsin. Rapid cycling refers to the short life cycle of 30 to 40 days, which was obtained by the Cooperative after interbreeding wild plants that were observed to possess a reduced reproductive time. Due to rapid cycling B. rapa's reduced reproductive period, the developmental stages of its ovules, were studied to identify any variations with it and the wild species (Smith, 1992). The conclusion of that study was that the qualitative stages were synonymous with the wild B. campestris, but that differences possibly existed based on size variations within the ovule stages (Smith, 92; Smith, 89). The re-examination of B. rapa was conducted to validate the findings of the previous study by Smith and to enhance it with quantitative data concerning the length and widths of the stages found as well as the ovary to identify any correlations that may exist. Whole flowers fixed in FPA 50 (5 ml formalin, 5 ml propionic acid, 90 ml 50 % ETOH) for a minimum of 24 hours Flowers were then transferred to 70 % ETOH for storage Flowers were dehydrated further by being placed into 80%, 85%, 90%, 95% and 100% ETOH for a minimum of 10 min each Inflorescences were then immersed into Herr Clearing Fluid (lactic acid, clove oil, chloral hydrate, phenol and xylene in a 2:2:2:2:1 ratio) for a minimum of 24 hours The ovaries were dissected from the flowers with the aid of a Nikon SMZ-U dissecting microscope; Images were also captured using a digital camera attachment The ovaries were placed onto Raj slides where further dissection to retrieve the ovules took place Ovules were then examined using a Nikon E600 microscope up to 1000x magnification and oil immersion techniques to identify and capture images using the digital camera attachment as well ImageJ software was utilized for measurement purposes which included the maximum length and width of the ovary (Fig. 1), ovule (Fig. 2) and developmental stages (Fig.3) Data was collected and analyzed using Microsoft Excel and SPSS software MATERIALS AND METHODS: Rapid Cycling B. rapa were grown using the Wisconsin Fast Plant growing system (Carolina Biological Catalog, Burlington, NC) Inflorescences collected at various sizes and ages starting after 15 days of initial planting RESULTS: The megaspore mother cell (mmc)(Fig. 4), dyad (Fig. 5) and tetrad (Fig. 6) stages of megasporogenesis were found The 2 nucleate (Fig. 7) and 4 nucleate (Fig. 8) stages of megagametogenesis were found but there were no 8 nucleate stages identified Using Pearson’s Coefficient, it was revealed that there was a less than 5 % correlation of ovary size compared to the developmental stages Growth patterns were observed when comparing the means (Tab. 1) and interquartile ranges (Fig. 9-11) CONCLUSIONS: The stages identified coincide with the stages of a polygnum type ovule development with the exception of being unable to identify the 8 nucleate stage. The 4 and 8 nucleate stages are often difficult to locate due to the nuclei existing in different planes. Therefore the qualitative data collected confirms the research conducted by Smith in There are trends associated with increasing ovary and ovule size as compared to what developmental stage the ovule is in, but Pearsons coefficient revealed a minimum correlation. This is most likely due to small sample sizes. FUTURE RESEARCH: Increase the population size measured to try and obtain a better correlation value Compare the quantitative embryological measurements of Brassica rapa to its wild variety as well as to other species within the genus. Compare embryological measurements within members of the Brassicaceae family and other families to identify any phylogenetic relationships that may exist qualitatively and quantitatively. LITERATURE CITED: Smith, Bruce B Comparisons of Early Ovule Development through Megagametogenesis in Rapid Cycling Brassica rapa L. and B. campestris L. Using the Herr Clearing Techniques. Angiosperm Pollen and Ovules. pp Smith, Bruce B. and Lightner, Jonathan E Early ovule development, megasporogenesis, and megagametogenesis in Brassica campestris L. (Cruciferae). Journal of the Pennsylvania Academy of Science. 63:3 pp ACKNOWLEDGEMENTS: Thank you to Dr. Bruce Smith for his assistance and guidance and to Dr. Jack Levisky for his tutorial. Figure 1. Longitudinal and lateral measurement of an ovary. Figure 2. Longitudinal and lateral measurement of an ovule. Figure 3. Longitudinal and lateral measurement of a stage. Figure 5. Dyad stageFigure 4. MMC stage Figure 6. Tetrad stageFigure 7. 2 Nucleate stage Figure 8. 4 Nucleate stageFigure 9. Interquartile range and medians for ovary area of each developmental stage Figure 10. Interquartile range and medians for ovule area of each developmental stage Figure 11. Interquartile range and medians for stage area of each developmental stage Stage n Mean Ovary Mean Ovule Mean Stage Size (mm 2) Size (mm 2 ) Size (mm 2 ) MMC x10 -5 Dyad x10 -5 Tetrad x Nuc Nuc Table 1. Means and population sample of each stage within the ovary, ovule and the stage itself.