Investigation and Comparison of Pistil Width in Relation to Early Ovule Development in Arabidopsis thaliana (L.)

Columbia EcotypeBy Bret Kilefner

gregorylab.bio.upenn.edu

Introduction:• Arabidopsis thaliana (L.) Heynh is a small flowering plant that is a member of the mustard family (Brassicaceae) (TAIR, 2008)

•A. thaliana (L.) Heynh is a model organism whose pistil length in Columbia ecotype has been analyzed, showing a direct correlation to its ovule development. Table 1 (Bowman Koorneef, 1994)

• There has not been a study of pistil width in relation to development of megasporogenesis through megagametogenesis of the ovule.

•Measurements of the pistil width will be correlated to the stages of ovule development to assist future research.

Hypothesis:I predict that pistil width will vary in all stages of ovule development in megasporogenesis through megagametogenesis in A. thaliana . These results will help future studies working with A. thaliana to save valuable time in determining the age of the flower.

Conclusion:My hypothesis was partially supported by my data. It was observed that pistil width varied significantly in ovules going through stages affiliated with megasporogenesis. However, pistil width was found to be non-significant in ovule stages associated with megagametogenesis.

Figure 1. Mean pistil widths +/- SD, categorized by the stages of megasporogenesis through megagametogenesis of the ovule. When compared across all subclasses, mean pistil width was significantly different (F(6,28) = 99.75, P=<0.0001). All pair-wise comparisons between subclasses were significant using a Tukey’s Multiple comparison post test, except comparisons between the last three stages of ovules. Non-significant pistil width are labeled (*) when ovule stage was compared to the succeeding subclass.

Table 1. (Bowman, Koorneef, 1994)

Collect floral buds from A. thaliana Columbia ecotype and place into FPA 50 fixative (min. 24 Hours)

Buds transferred from fixative and then taken through a dehydration series using EtOH

Pistils were removed and widths measured, then ovules were removed and assessed to determine stage of development.

The Buds were transferred to Herr’s Clearing Fluid (min. 24 Hours)

Pictures were saved using the Nikon Microscopes and Digital Cameras on Nikon Elements Version 4.0 Software

Means of pistil width were calculated assessed using a One-Way Anova with Tukey's multiple comparison post-test

Methods

Results: MMC Dyad Tetrad

2-Nucleate 4-Nucleate 8-Nucleate

AcknowledgmentsI would like to thank Dr. Bruce Smith for his help and support throughout this research, as well as Shane Lohss for teaching me the required techniques for this analysis.

Table 1. Stages of embryo sac development in A. thaliana Columbia ecotypeApproximate floral stage

Stage of embryo sac development

Ovule morphology

Pistil Length (mm)

9 Ovule primordia arise0.15-0.4

10 Ovule primordia elongate 0.4-0.5

11 Megasporocyte Inner and outer integuments initiated 0.5-1.0

4 megaspores

Functional megaspore (1-nucleate embryo sac) 1.0-1.5

Early 12 2-nucleate embryo sac

Funiculus and nucellus curve; other integuments exhibits assymmetric growth

1.5-2.0

Mid 12 4-nucleate embryo sacInteguments grow upward around nucellus

Late 12 8-nucleate embryo sac

Outer integuments begins to cover both inner integuments and nucellus

13 Mature embryo sac (8-nucleate, 7-celled)

Integuments envelop nucellus; micropyle positioned near funiculus

2.0-2.5

14 Fertilization Embryo sac becomes increasingly curved 2.5-3.0

0

100

200

300

400

Mean Pistil Width vs. VariousStages of Ovule Development

Ovule Stage

Pis

til W

idth

(µm

) * * *

Literature Cited:Bowman, J., Koornneef, M. (1994). Arabidopsis: An Atlas of Morphology and Development. New York: Springer

Gasser, C.S., Robinson-Beers, K. (1993). Pistil Development. American Society of Plant Physiologists. 5(10). Pp 1231-1239.

Robbelon G. (1965). The Laibach standard collection of natural races of Arabidopsis. Inf. Serv. 2: 36-47.

Smith, 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

Smith, B., Herr, J 1971. Ovule Development, Megagametogeneisis and Early Embryogeny in Ammannia Coccine Rothb.. . . The Journal of the Elisha Mitchell Scientific Society. 87(4.

TAIR, (2008). About Arabidopsis. TAIR- Home Page. Retrieved November 19, 2011 on the world wide web: http://www.arabidopsis.org/portals/education/aboutarabidopsis.jsp

.