L I J Selected Stages of Ovule and Anther Development in Brassica oleracea L. Benjamin Rogers, Department of Biological Sciences, York College of Pennsylvania Abstract Various varieties of wild Brassica oleraceae are vegetables well known to all and include, but not limited to, cabbage and broccoli. Floral buds were prepared for the study through a series of processes to fix, dehydrate, and clear the tissue. With this technique it is possible to study microscopic growth of plants through several cell layers. Maheshwari, Herr, and others, in the mid to late 20th century, have provided evidence for plant systematics that have focused on qualitative and quantitative gynoecial and androecial development in angiosperms. Ovule development and anther development has not been reported for Brassica oleracea L., nor has it been reported for the rapid- cycling variety. Selected stages of ovule and anther development in Rapid cycling B. oleracea L. are presented in this study. Planting, Growing, Harvesting Obtain rapid cycling (rcBR), Brassica oleracea seeds Grow according to the Wisconsin Fast Plants Manual Collect floral buds Collections fixed in Formalin, Propionic acid, Ethyl alcohol 50%, (aka FPA50) for a minimum of 24 hours After fixing, the dehydration process can start Methods Dehydration and Clearing 70%ETOH 10min 80%ETOH 10min 90%ETOH 10min 95%ETOH 10min 100%ETOH 10min Clearing Fluid Acknowledgements Bruce Smith, Ph.D., mentor Introduction The B. oleracea in this study is the Rapid Cycling variety. This plant will grow to over 20cm, and has yellow and white flowers. To study the growth and development of plants a clearing technique devised by J.M. Herr is very helpful (Herr 1971). This technique clears botanical tissue thus allowing images of cellular activity to be seen at various focal planes. For the last century ovule and anther development has been studied by the likes of Maheshwari, Herr, and others. Earlier studies stressed the use of microscopes with drawing tubes and cellular images were traced with a pencil and this documented the stages of ovule development. In the past few decades, photographs replaced the drawing tube and recently digital images are the standard for capturing cellular imagery. Ovule development and anther development has not been reported for the wild type nor the rapid cycling varieties of B. oleracea. Selected stages of ovule and anther development in Rapid cycling B. oleracea L are presented in this study using Nikon phase contrast microscopes and a Nikon digital camera to capture digital images of the stages reported. Figure Legend* A. Young ovule with outer and inner integuments starting to form and the archesporial cell B. Tetrad formation C. Tetrad with Chalazal ( C1 and C2) becoming dominant over the two spores toward the micropylar end. D. Two-nucleate stage E. Chalazal spore in nucellus F. Ovule showing inner integument to be approx. 2 cell layers thick, and outer integument 3+ layers thick G. Young ovule with (1)outer, (2)inner integuments, and (3)nucellus H. Ovule with funiculus attached to placenta I. Microspore(s) contained in a portion of sporogenous anther tissue J.Squashed anther tissue with Dyad formation K. Tetrad in tetrahedral position L. Pollen grain with arrow pointing to exine layer *all images at 1000x (oil) except for G (400x) and H (10x) Literature Herr, J.M. Jr A new clearing-squash technique for the study of ovule development in angiosperms. American Journal of Botany.58: Smith, Bruce and M.M. Virden Early Ovule Development, Megasporogenesis, and Megagametogenesis in Rapid Cycling Brassica nigra (L) Koch. Journal of the Pennsylvania Academy of Science. 73(3): Discussion The images collected from B. oleracea show that the ovule development is similar to previous studies of other Brassica species such as epitropous ovules and survival of chalazal spore for megagametophytic development (Smith and Virden, 1999). Mature ovules becoming campylotropous, the number of cells in the inner and outer integument and the lack of multiple megaspore mother cells are all novel findings. The data given represent selected gynoecial and androecial stages, which thus far have not been reported. With further study of this commercially important species, more could be learned about its ovule and anther development. Results Young ovules show nucellar tissue with archesporial cell and young integumetary buds (Fig.A). A linear tetrad is fully formed when the ovule begins to form an epitropous position (Fig. B). The two spores toward the chalazal end begin to grow and crush the two micropylar spores (Fig. C). The spore nearest the chalazal end begins the mitotic divisions forming a 2-nucleate megagametophyte (Figs. D and E). The outer integument has at least three cell layers and the inner integument has two cell layers (Fig. F). Young ovule with outer and inner integuments beginning to swell around the protruding nucellus (Fig. G). Whole ovule attached to a partial placenta beginning to form into the epitropous and campylotropous ovule position (Fig. H). Sporogenous tissue of the anther develops into a dyad and later a tetrahedral tetrad before finally becoming a mature pollen grain (Figs. I,J,K.L). K A 1 2 B C D F II OI G H E