1. Environmental Effects on the size of the Female Megagametophyte Stages in two Arabidopsis thaliana ecotypes Olivia Gross and Bruce Smith York College of Pennsylvania Methods Results Figure 1. Fibonacci Sequence analysis using mean width and length for Libya 2-nucleate stage Figure 2. Fibonacci Sequence analysis using mean width and length for Canary Islands 8-nucleate stage Objectives Conclusion Literature Cited A – Functional Megaspore (Libya), B – 2-nucleate (Libya) C – 8-nucleate (Libya), D – 2-nucleate (Canary Islands) Introduction B A CD Arabidopsis thaliana is a small flowering plant belonging to the Brassicaceae family. While of little agronomic importance, A. thaliana has become an essential tool in plant biology, as well as genetic and molecular research. There are nearly 800 ecotypes of A. thaliana found all over the world. They available through Ohio State University’s seed stock center, Arabidopsis Biological Research Center (ABRC 2009). Megagametogenesis is the development of a megaspore into an embryonic sac. The four stages being researched are the functional megaspore, 2-nucleate, 4-nucleate, and 8- nucleate. Previous research observed ovule development in megagametophytic stages of 5 species of Cornus L. using a new clearing technique (Smith 1975). Differences between species during this stage resulted in the suggested removal of C. florida from the genus Cornus. Similar research is necessary for the many different ecotypes of Arabidopsis thaliana. Investigate early ovule development through four stages of megagametogenesis in two ecotypes of Arabidopsis thaliana: CS1064 from an elevation of 1000 meters in the Canary Islands and CS1380 from an elevation of 250 meters in Libya. Ovule width and length measurements will be taken from each of the four stages. Data will be analyzed using the Fibonacci Sequence to determine if there are differences between the two ecotypes. It is hypothesized that the divergent environments of the two ecotypes will not affect the size of the female megagametophyte stages. 8 wells of each ecotype planted and placed in cold room for 5-7 days CS1064 – Canary Islands CS1380 – Libya After cold treatment, plants placed under lights until inflorescences appeared Inflorescences were removed and placed in FPA50 for a minimum of 24 hours After removal from the FPA50, inflorescences dehydrated to 100% EtOH Inflorescences continued to be removed from growing flowers and placed in FPA50 Dehydrated inflorescences placed in Herr fluid for 24 hours minimum before being viewed under microscope Ovules dissected from pistil measured and recorded (minimum of 10 measurements per ecotype) WIDTH: 8.73 LENGTH: 20.32 2.327605956 1 29.05 1.429625984 2 49.37 1.699483649 3 78.42 1.588414017 4 127.79 1.629558786 5 206.21 1.613663041 6 340.00 1.619708065 7 540.21 1.61739651 8 874.21 1.617278077 9 1414.42 1.617940769 10 2288.63 1.618069597 11 3703.05 1.618020388 12 5991.68 1.618039184 13 9694.73 1.618932004 14 15686.41 1.618034747 15 25381.14 1.618033699 16 41067.55 1.618034099 17 66448.69 1.618033947 18 WIDTH : 14.33 LENGTH: 54.98 3.836706211 1 69.31 1.260640233 2 124.29 1.793247728 3 193.60 1.557647437 4 317.89 1.641993802 5 511.49 1.609015697 6 829.38 1.621497977 7 1340.87 1.61671369 8 2170.25 1.61683871 9 3511.12 1.617841263 10 5681.37 1.618107612 11 9192.49 1.618005868 12 14873.86 1.61804473 13 24066.35 1.618029886 14 38940.21 1.618035556 15 63006.56 1.61803339 16 101946.77 1.618034217 17 164953.33 1.618033901 18 Fibonacci Sequence Results The 3-decimal phi constant, 1.618, and the 7-decimal phi constant, 1.6180339, were reached at the 11 th and 18 th divisions, respectively, for all four stages in both ecotypes. It is concluded that the divergent environments of Libya and the Canary Islands did not affect the size of the female gametophyte. Similar research needs to be conducted for all ecotypes of Arabidopsis thaliana to further research the relationship between environment and megagametophyte stages. ABRC 2009. The Ohio State University. Available from: http://www.abrc.osu.edu/. Smith, B.B. 1973. The use of a new clearing technique for the study of early ovule development, megasporogenesis,and megagametogensis in five species of Cornus L. American Journal of Botany 60(4): 322-338. Smith, B.B. 1975. A quantitative analysis of the megagametophyte of five species of Cornus L. American Journal of Botany 62(4): 387-394. I would like to thank Dr. Smith for his constant support and guidance throughout the senior thesis project.