1. Is there an ideal pistil length in which each stage of Megagametogenesis can be observed in Arabidopsis thaliana? Introduction: Comparing the haploid stages of seed development provides yet another morphological feature that can be used to determine relationships between and among species by phylogenists. These stages, although difficult to isolate and quantify, can now be studied using the Herr Clearing techniques (Smith 1973). Ecotypes are members of the same species that appear differently when viewed in varying environmental conditions, i.e., Arabidopsis thaliana (Rédei, 1969). Arabidopsis thaliana with over 800 ecotypes identified is a good research model because its developmental stages are easily discernible, it’s inexpensive, easy to order, and it goes from seed to seed in less than a month. I chose to compare three ecotypes looking carefully at the sizes of their pistils and the megagametophytic stages; functional megaspore, 2-nucleate, 4-nucleate, and 8-nucleate stages to see if a single pistil length might contain each of these megagametophytic stages. Objectives: Compare pistil lengths of 3 ecotypes against the megagametophytic stages they contain (Webb & Bowman, 1994). Joy Haring Department of Biological Sciences, York College of Pennsylvania Definitions: Megagametogenesis : the mitotic divisions of a megaspore mother cell that later develops into an embryo sac, all of which is the megagametophyte. Ecotype: genetic variety within a species that arises as a result of adaptions to the local environment. For Arabidopsis thaliana, 800+ ecotypes are known. Pistil: the female reproductive parts of the flower. The pistil is divided into the stigma, style and ovary. The ovary is composed of individual ovules. Haploid: the number of chromosomes that represents ½ the diploid number. Functional megaspore: after meiotic division four cells are formed that are haploid and three of them die. The remaining one is the functional megaspore. Ovules: the structure attached to the placenta of the ovary and eventually becomes a seed. Table 2. Comparison of Ovary lengths to number of ovules in each developmental stage. Length of the Pistil in mm # ovules FM a # ovules 2- nucleate # ovules 4- nucleate # ovules 8- nucleate Ovary 1 (Le) b 1.5 Ovary 2 (Le)2.13 Ovary 3 (Le)21 Ovary 4 (Le)2110 Ovary 5 (Le)17 Ovary 6 (Col) c 1.4 Ovary 7 (Le)1.13 Ovary 8 (Le)1.81111 Ovary 9 (Col)1.3 Ovary 10 (Le)1.211 Ovary 11 (D) d 1.16 Ovary 12 (D)1.112 Ovary 13 (D)18 Ovary 14 (D)1.9133 a Functional Megaspore b Le: Landsberg erecta c Col: Columbia d D: Dijon Future Studies: To work with other ecotypes to determine if there is a universal pistil length or if there is variation within the ecotype. To compose a table that compares floral stages, appearance of the flower, anther length and its morphology. To determine ideal pistil lengths in other species and create a table for them modeled after Webb and Bowman (1994). Literature Cited: Rédei, G.P. 1969. Arabidopsis thaliana (L.) Heynh. A Review of the Genetics and Biology. Bibliographia Genetica. 21: 1-151. Smith, B.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. American Journal of Botany.60 (4): 322-338. Webb, M.C. and Bowman, J.L. 1994. Arabidopsis: An Atlas ofMorphology and Development. Springer-Verlag, New York. Acknowledgements: I would like to thank Dr. Smith for his continued expertise, guidance and understanding through the entire Senior Thesis process, and throughout the Biology curriculum. Grow multiple wells of each ecotype At first sign of flowering remove inflorescence and place in fixative (FPA 50 Minimum 24 hrs.) Continue collections and fixing until some flowers go to fruit Place flowers in dehydration series, 70% to 80% to 85% to 90% to 95% to 100% EtOH Minimum 10 mins. in each concentration Herr Fluid Measure pistil length View dissected ovule with phase contrast microscope Conclusion: One ovary from the Landsberg erecta ecotype was found to have ovules in each stage of development. Another ovary from Landsberg erecta has ovules in two of the stages, indicating more ovaries with pistil lengths in between these two could have more stages. From this small sample size, an ideal pistil length cannot be definitely determined. Methods: 4-nucleate stage: 8-nucleate stage: Whole Pistil Functional Megaspore 2-nucleate stage Taken from Webb and Bowman, 1994 Table 1. Stages of embryo sac and ovule development Approximate floral stage Stage of embryo sac development Ovule morphology Pistil length (mm) Appearance of flower 9Ovule primordia arise 0.15-0.4 10Ovule primordia elongate 0.4-0.5 11Megasporocyte 4 megaspores Functional megaspore Inner and outer integuments initiated 0.5-1.0 1.0-1.5 Early 122-nucleate embryo sac Funiculus and nucellus curve; other integument exhibits asymmetric growth 1.5-2.0 Mid 124-nucleate embryo sac Integuments grow upwards around nucellus Late 128-nucleate embryo sac Outer integument begins to cover both inner integument and nucellus Results: